Section A7.1.1.1.1 Hydrolysis as a function of pH and...

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 February 2004

RCC project no. 852224 of 10

Section A7.1.1.1.1 Annex Point IIA7.6.2.1

Hydrolysis as a function of pH and identification of

breakdown products

1 REFERENCE

Official use only

1.1 Reference Lewis, C.J. (2000): (14

C)-TI-435: Hydrolytic stability.

Covance, Harrogate, England; unpublished report no. 586/140-D2142

1.2 Data protection Yes

1.2.1 Data owner Sumitomo Chemical Takeda Agro Co., Ltd.

1.2.2 Companies with

letter of access

None

1.2.3 Criteria for data

protection

Data on existing a.s. submitted for the first time for entry into Annex I.

2 GUIDELINES AND QUALITY ASSURANCE

2.1 Guideline study Yes

92/69/EEC, C.7 and US EPA, Subdivision N, Section 161-1

2.2 GLP Yes

2.3 Deviations None (to EC directive)

3 MATERIALS AND METHODS

3.1 Test material [thiazolyl-14

C]TI-435

3.1.1 Lot/Batch number A-980501

3.1.2 Specific activity 3.88 MBq/mg

968.76 MBq/mmol

3.1.3 Purity Radiochemical purity: >98%

X

3.1.4 Further relevant

properties

Water solubility (20°C): 327 mg/L

(see Morrissey & Kramer, 2000a)

3.2 Reference substance Yes (for identification of parent compound and degradation products):

TI-435 (unlabelled parent compound)

Degradation products:

CTNU: N-(2-chlorothiazol-5-ylmethyl)-N’-nitrourea

TZMU: N-(2-chlorothiazol-5-ylmethyl)-N’-methylurea

ACT.HCl: 2-chlorothiazol-5-ylmethylamine hydrochloride

3.3 Test solution See Tables A7_1_1_1_1-1 and A7_1_1_1_1-2 X

3.4 Testing procedure

3.4.1 Test system See Table A7_1_1_1_1-3

3.4.2 Temperature Preliminary study: 50°C (pH 4, 7 and 9)

Definitive study: 25°C (pH 5, 7 and 9)

Additional definitive study: 62°C and 74°C (pH 9)

3.4.3 pH All vials tested were within +/- 0.2 pH units of the intended pH both

after sterilization and at the end of the experiment.

3.4.4 Duration of the test Preliminary study: 5 days (pH 4 and 7), 25 days (pH 9)

Definitive study: 33 days (pH 5, 7 and 9)

Additional definitive study (pH 9): 7 days (62°C) and 1.9 days (74°C)

3.4.5 Number of replicates As given in Table A7_1_1_1_1-2





breakdown products

3.4.6 Sampling Sampling intervals:

Preliminary study: 0, 0.1, 1 and 5 days (pH 4 and 7) and 0, 0.1, 1, 5, 11,

15, 19 and 25 days (pH 9)

Definitive study: 0, 5, 9, 15, 20, 27 and 33 days (pH 5, 7 and 9)

Additional definitive study (pH 9): 0, 1, 3 and 7 days (62°C) and 0,

0.25, 1 and 1.9 days (74°C)

3.4.7 Analytical methods Identification of the parent compound and transformation products:

HPLC (ODS-H column, UV detection at 265 nm) or TLC (silica gel

K6F plates).

Measurement of radioactivity: LSC

No sample preparation was necessary.

3.5 Preliminary test Yes,

4 RESULTS

4.1 Concentration and

hydrolysis values

See Table A7_1_1_1_1-4 to Table A7_1_1_1_1-11

4.2 Hydrolysis rate

constant

See Table A7_1_1_1_1-12

4.3 Dissipation time See Table A7_1_1_1_1-12

The half-life at 20°C and pH 9 was calculated from the Arrhenius plot.

The calculated rate constant was 0.000494 day-1

and from this a half-life

of 1401 days (ca. 3.8 years) was calculated.

4.4 Concentration time

data

See Figure A7_1_1_1_1-1 to A7_1_1_1_1-3

Since TI-435 was stable at pH 4, 5 and 7 (25°C and 50°C) and at pH 9

(25°C) no concentration-time plot is provided.

4.5 Specification of the

transformation

products

See Table A7_1_1_1_1-13

No transformation products were found at pH 4, 5 and 7 (25°C and

50°C) and only trace amounts were found at pH 9 (25°C).

Figure A7_1_1_1_1-4 shows the Proposed hydrolysis pathways of

TI-435.

5 APPLICANT'S SUMMARY AND CONCLUSION

5.1 Materials and

methods

Degradation – abiotic degradation hydrolysis as a function of pH:

92/69/EEC, C.7 and US EPA, Subdivision N, Section 161-1; Deviations

(to EC directive): none

5.2 Results and

discussion

The radiochemical purity of the application solution was determined at

each time of dosing (preliminary, definitive and additional definitive

study) and was at any time >98%. The results of the study are

summarised in Tables A7_1_1_1_1-4 to A7_1_1_1_1-11. The half-lives

were calculated using first-order kinetics. The total recovery of the

applied radioactivity was in the range of 96% to 103%.

A half-life of 1401 days for a temperature of 20°C and a pH-value of 9

was calculated according to the Arrhenius equation using the data from

the tests performed at 50°C, 62°C and 74°C.

5.2.1 kH pH 9: 0.048 (50°C), 0.188 (62°C), 1.013 (74°C)

5.2.2 DT50 pH 9: 14.4 days (50°C), 3.7 days (62°C), 0.68 days (74°C)

5.2.3 r2

pH 9: 0.997 (50°C, 62°C and 74°C)

5.3 Conclusion TI-435 is stable in sterile buffer solutions at pH 4, 5 and 7, but degrades





breakdown products

at pH 9. However, at relevant temperatures of 20°C the degradation is

very slow (calculated half-life: 1401 days). Relevant amounts of

metabolites were formed only at elevated temperatures.

5.3.1 Reliability 1

5.3.2 Deficiencies None

Evaluation by Competent Authorities

EVALUATION BY RAPPORTEUR MEMBER STATE

Date 2004/11/20

Materials and Methods Despite minor deficiencies applicant’s version is acceptable.

Comments:

The mentioned radiochemical purity of > 98% of test substance [thiazolyl-14

C]TI-

435 corresponds to the radiochemical purity that was determined in control

measurements within the study time. The stated radiochemical purity according to

the certificate of analysis amounts to 99.2 % (TLC radiochromatography) and

99.4 % (HPLC radiochromatography), respectively.

The correct caption of Table A_7_1_1_1_1-2 is “Description of test solution”

instead of the current caption “Type and composition of buffer solution”.

The description for the preparation of the test medium (see Table A_7_1_1_1_1-

2) should be more comprehensible as follows: “Aliquots of 3 mL were taken from

the buffer solutions and were dispensed into borosilicate glass vials which were

then sealed and sterilised by autoclaving. (…)”

Results and discussion Despite minor deficiencies applicant’s version is acceptable.

Comments:

The sampling times of the hydrolysis of a.s. in pH 9 at 74 °C in Table

A_7_1_1_1_1-11 are 0, 0.25, 1, and 1.9 days and not 0, 1, 3, and 7 days.

The hydrolysis rate constant kh in Table A_7_1_1_1_1-12 is listed in the unit

[days-1

]. The given justification that no hydrolysis rate constants at pH 4, 5, and 7

can be calculated (see *) is not appropriate. A rate constant can not be calculated

as there is no significant hydrolysis. The first-order reaction model can not fit the

data as the model is inadequate to describe the data (see low correlation

coefficients r2).

The duration of the additional definitive test in pH 9 at 74 °C in Table

A_7_1_1_1_1-12 is 1.8 days which is more precise

In the original study at Doc. IV-A, Section No. 7.1.1.1.1 the Arrhenius equation

and the calculation procedure of rate constant in pH 9 at 20 °C is lacking. Only

the Arrhenius plot with intercept and slope is pictured. Generally, the whole

calculation procedure should be demonstrated in the study reporting to be

transparent.

Conclusion Applicant’s version is acceptable.

Comments:

The hydrolysis rate constant kh under item 5.2.1 is listed in the unit [days-1

].

The degradation products in pH 9 are CTNU (N-(2-chlorothiazol-5-ylmethyl)-N’-

nitrourea), TZMU (N-(2-chlorothiazol-5-ylmethyl)-N’-methylurea), and ACT.HCl

(2-chlorothiazol-5-ylmethylamine hydrochloride). The latter seems to be the final

transformation product.

Reliability 1





breakdown products

Acceptability Original study and study summary are acceptable

Remarks

COMMENTS FROM

Date Give date of the comments submitted

Materials and Methods Discuss additional relevant discrepancies referring to the (sub)heading numbers

and to applicant's summary and conclusion.

Discuss if deviating from view of rapporteur member state

Results and discussion Discuss if deviating from view of rapporteur member state

Conclusion Discuss if deviating from view of rapporteur member state

Reliability Discuss if deviating from view of rapporteur member state

Acceptability Discuss if deviating from view of rapporteur member state

Table A7_1_1_1_1-1: Type and composition of buffer solutions

pH Type of buffer (final

molarity)

Composition

4 0.01 M potassium hydrogen

phthalate

0.01 M sodium hydroxide solution were added to 0.01 M

potassium hydrogen phthalate solution until the pH was

4.0 at 50°C.

5 0.01 M sodium citrate 0.01 M trisodium citrate solution were added to 0.01 M

citric acid solution until the pH was 5.0 at 25°C.

7 0.01 M TRIS maleic acid 0.01 M sodium hydroxide solution were added to 0.01 M

TRIS-maleic acide solution until the pH was 7.0 (at 25°C

or 50°C).

9 0.01 M borate-boric acid The pH of 0.0025 M sodium tetraborate solution was

adjusted to 9.0 with either 0.01 sodium hydroxide solution

or 0.01 boric acid solution (at 25°C, 50°C, 62°C or 74°C).



Table A7_1_1_1_1-2: Type and composition of buffer solutions

Criteria Details

Purity of water BDH ‘HiPerSolv’ water for HPLC (Merck)

Preparation of test medium Aliquots of 3 mL were dispensed into borosilicate glass

vials which were then sealed and sterilised by

autoclaving. About 0.9 µg [thiazolyl-14

C]TI-435

diluted in approximately 20 µl acetonitrile (equivalent

to 45 mg/L) was aseptically injected onto the surface of

the buffer solutions and then mixed.

Test concentrations (mg a.i./L) 0.3 mg a.i./L

Temperature (°C) pH 4: 50°C

pH 5: 25°C

pH 7: 25°C/50°C

pH 9: 25°C/50°C/62°C/74°C

Controls Two control vials per pH-value

Identity and concentration of co-solvent Acetonitrile, concentration: ca. 0.7%

Replicates Duplicates

Table A7_1_1_1_1-3: Description of test system

Criteria Details

Glass ware Borosilicate glass vials (capacity: 4 mL, type 4-CV

from Chromacol, Welwyn Garden City, Hertfordshire)

Other equipment Water bath, pH meter

Method of sterilization Autoclaving

Table A7_1_1_1_1-4: Hydrolysis of [14

C]TI-435 and transformation products in pH 5 at 25°C (expressed

as percentage of applied radioactivity)

TI-435

Sampling times (days)

0 5 9 15 20 27 33

Parent compound 98.3 98.1 98.4 97.1 97.0 96.5 99.1

ACT.HCl ND ND ND ND ND ND ND

CTNU ND ND ND ND ND ND ND

TZMU ND ND ND ND ND ND ND

Unresolved ND 0.2 0.3 0.2 0.3 0.5 0.7

Total % recovery 98.3 98.3 98.7 97.3 97.3 96.9 99.8

ND = Not detected






TI-435


0 5 9 15 20 27 33

Parent compound 98.1 99.3 97.5 97.7 97.1 102.9 98.1


CTNU ND ND ND ND ND ND ND

TZMU ND ND ND ND ND ND ND

Unresolved 0.3 0.2 0.9 0.8 0.7 0.4 0.3

Total % recovery 98.5 99.6 98.4 98.5 97.8 103.4 98.4

ND = Not detected




TI-435


0 5 9 15 20 27 33

Parent compound 97.7 98.2 99.8 97.8 99.2 96.4 93.8


CTNU ND ND ND ND 0.9 1.4 1.8

TZMU ND ND ND ND 0.3 0.4 0.6

Unresolved 0.3 0.3 0.8 0.1 0.4 0.5 0.6

Total % recovery 98.0 98.6 100.6 97.9 100.9 98.8 96.8

ND = Not detected






TI-435


0 0.1 1 5

Parent compound 100.7 96.3 99.1 97.5

ACT.HCl ND ND ND ND

CTNU ND ND ND ND

TZMU ND ND ND ND

Unresolved 0.5 0.6 0.7 0.3

Total % recovery 101.2 96.9 99.8 97.8

ND = Not detected




TI-435


0 0.1 1 5

Parent compound 100.6 96.2 100.4 95.5

ACT.HCl ND ND ND ND

CTNU ND ND ND ND

TZMU ND ND ND ND

Unresolved 0.8 0.6 0.6 06

Total % recovery 101.4 96.8 101.1 96.1

ND = Not detected






TI-435


0 0.1 1 5 11 15 19 25

Parent compound 98.9 100.1 95.6 77.9 57.6 50.0 40.9 29.1

ACT.HCl ND ND 0.4 10.4 27.5 36.3 44.3 52.8

CTNU ND ND 3.3 8.2 7.7 6.0 4.9 3.9

TZMU ND ND 1.3 3.2 6.0 7.7 9.4 10.8

Unresolved 0.5 0.4 0.8 0.3 0.6 0.2 0.4 0.7

Total % recovery 99.5 100.5 101.5 100.1 99.3 100.2 99.9 97.3

ND = Not detected




TI-435


0 1 3 7

Parent compound 99.6 83.0 55.8 27.1

ACT.HCl ND 7.7 28.0 53.5

CTNU ND 5.1 3.1 1.7

TZMU ND 3.2 8.2 14.7

Unresolved 0.4 0.6 0.4 0.7

Total % recovery 100.0 99.6 95.6 97.7

ND = Not detected






TI-435


0 1 3 7

Parent compound 99.5 74.6 36.6 14.4

ACT.HCl ND 10.8 41.2 59.2

CTNU ND 4.7 2.1 0.7

TZMU ND 6.5 16.8 22.6

Unresolved 0.5 0.4 0.6 0.5

Total % recovery 100.0 97.0 97.3 97.3

ND = Not detected

Table A7_1_1_1_1-12: Hydrolysis rate constant as a function of pH and temperature

Study pH Temperature

[°C]

Duration

[days]

kh Half-life

(days)

Correlation

coefficient

Preliminary

test

4 50 5 0.003* * 0.075*

7 50 5 0.007* * 0.290*

9 50 25 0.048 14.4 0.997

Definitive

test

5 25 33 0.000* * 0.008*

7 25 33 -0.001* * 0.071*

9 25 33 0.001 * 0.210*

Additional

definitive

test

9 62 7 0.188 3.7 0.997

9 74 <2 1.013 0.68 0.997

* Rate of degradation too slow to compute a half-life



Table A7_1_1_1_1-13: Specification and amount of transformation products

Code

name

CAS name Maximum amount (percentage of applied radioactivity) in

pH 9 aqueous solution at

50°C 62°C 74°C

ACT.HCl 2-chlorothiazol-5-ylmethylamine

hydrochloride

52.8 53.5 59.2

CTNU N-(2-chlorothiazol-5-ylmethyl)-

N’-nitrourea

8.2 5.1 4.7

TZMU N-(2-chlorothiazol-5-ylmethyl)-

N’-methylurea

10.8 14.7 22.6

Figure A7_1_1_1_1-1: Hydrolysis of [14C]TI-435 and transformation products in pH 9 at 50°C



Figure A7_1_1_1_1-4: Proposed hydrolysis pathways of TI-435


RCC project 852224 Page 1 of 7


Phototransformation in water including identity of the

products of transformation

1 REFERENCE Official

use only

1.1 Reference Babczinski, P. and Bornatsch, W. (2000): Photolysis of [nitroimino-14

C]TI-435 and [thiazolyl-2-14

C]TI-435 in sterile aqueous buffer

solution.

Bayer AG, 51368 Leverkusen, Germany; unpublished report no. MR

248/00




letter of access

None


protection




SETAC and US EPA 161-2

2.2 GLP Yes

2.3 Deviations Yes

Deviations (to SETAC): temperature 25±1°C instead of 20±3°C


3.1 Test material [14

C]TI-435

3.1.1 Radiolabelling [nitroimino-14


C]TI-435

3.1.2 Lot/Batch number [nitroimino-14

C]TI-435: 11553/1

[thiazolyl-2-14

C]TI-435: 11649/2

3.1.3 Specific

radioactivity

[nitroimino-14

C]TI-435: 3.78 MBq/mg

[thiazolyl-2-14

C]TI-435: 3.84 MBq/mg

3.1.4 Purity [nitroimino-14

C]TI-435:

Radiochemical purity: > 99% according to radio HPLC

Chemical purity: > 99% according to HPLC, UV at 210 nm

[thiazolyl-2-14

C]TI-435:

Radiochemical purity: > 99% according to radio TLC


3.1.5 UV/VIS absorption

spectra and

absorbance value

See Figure A7_1_1_1_2-1


properties








3.2 Reference

substances

Yes (for identification of parent compound and degradation products):



HMIO: 4-Hydroxy-2-methylamino-2-imidazolin-5-one

MAI: 3-Methylamino-1H-imidazo[1,5-c]imidazole

MG: Methylguanidine

MIO: 2-Methylamino-2-imidazolin-5-one

MU: Methylurea

TMG: N-(2-chlorothiazol-5-ylmethyl)-N’-methylguanidine


FA: Formamide

3.3 Test solution See Table A7_1_1_1_2-1



3.4.2 Properties of light

source

See Table A7_1_1_1_2-2

3.4.3 Determination of

irradiance

The measurement of the intensity of artificial irradiance was performed

with a radiometer (see Table A7_1_1_1_2-2) and by actinometry (see

Table A7_1_1_1_2-1).

3.4.4 Temperature 25±1°C

3.4.5 pH The initial and the final pH was 7.0 for both the nitroimino-14

C]TI-435

solution and the [thiazolyl-2-14

C]TI-435 solution (test solutions and dark

controls).

3.4.6 Duration of the test 18 days

3.4.7 Number of

replicates

One test vessel per radiolabel. Initial test volume: 200 mL.

3.4.8 Sampling Directly after transferring the test solution into the test vessel, an aliquot

of 20 mL was withdrawn for set-up of the dark controls (3 x 5 mL) and

as zero-time samples (3 x 1 mL). Thereafter, volumes of 4 mL were

sampled at 1.5, 4, 24, 120, 264 and 432 hours.

One mL of the 4 mL samples was frozen immediately after sampling as a

reserve material.

3.4.9 Analytical methods Radioactivity: liquid scintillation counting (LSC)

Concentration of test substance and photolysis products: TLC on RP-18

plates and silica gel plates.

LOQ for a single zone: 0.5 Bq (corresponding to ca. 0.43% of the

applied radioactivity for a single spot on a two-dimensional TLC plate).

Photodegradates were purified by HPLC using an HP 1050 Liquid

Chromatograph with UV detector and radioactivity monitor, Ramona-90

with a solid glass scintillator cell.

The test solutions were analysed directly without sample preparation.

The PU foam plug of each trap was extracted twice with 20 mL ethyl

acetate. Aliquots of the extract were used for radioactivity assessment

and were further investigated if the radioactivity content was > 1% of the

applied amount.

The soda lime of the CO2 trap was dissolved in 18% HCl. The liberated

CO2 was absorbed with Carbosorb/Permafluor E + and the radioactivity






was determined by LSC.

3.5 Transformation

products

Transformation products tested: Yes

3.5.1 Method of analysis

for transformation

products

As described in Section 3.4.9.

4 RESULTS

4.1 Screening test Not performed

4.2 Actinometer data Actinometry was only used to measure the intensity of the artificial

irradiance (photon flow: 1.89 x 1017

photons/s, photon flow density:

125.86 x 1014

photons/s x cm2).

4.3 Controls [nitroimino-14

C]TI-435: C0 = 100.1% AR, Cend = 93.8% AR

[thiazolyl-2-14

C]TI-435: C0 = 95.0% AR, Cend = 104.1% AR

4.4 Photolysis data

4.4.1 Concentration

values


4.4.2 Mass balance See Table A7_1_1_1_2-3

4.4.3 kcp No actinometer study

4.4.4 Kinetic order pseudo first order

4.4.5 kcp / k

ap No actinometer study

4.4.6 Reaction quantum

yield (cE)

No actinometer study

4.4.7 kpE No actinometer study

4.4.8 Half-life (t1/2E) Experimental half-life: 3.3 hours (mean of two radiolabel studies).

Corresponding half-life under summer (June) solar light conditions at

Phoenix/USA: 0.6 days.

Corresponding half-life under winter (December) solar light conditions

at Phoenix/USA: 1.6 days.

x

4.5 Specification of

the transformation

products



5.1 Materials and

methods

Test guidelines: SETAC and US EPA 161-2

Deviations (to SETAC): temperature 25±1°C instead of 20±3°C

The photolytic degradation of [nitroimino-14


C]TI-435 was investigated in sterile aqueous buffer solution at pH 7.

The test solutions were exposed to simulated sunlight in 350 mL quartz

glass vessels in the Heraeus ®Suntest apparatus equipped with a xenon

lamp and a cooling platform. Dark controls were kept in a second

Suntest apparatus. The samples were maintained at 25±1°C and exposed

to light for 18 days.






5.2 Results and

discussion

TI-435 degraded rapidly in the irradiated samples with a mean DT50-

value (two radiolabels) of 3.3 hours.

Major photolytic degradation products of [nitroimino-14

C]TI-435 were

MG, TZMU, HMIO and MU with maximum levels of 34.7%, 29.3%,

26.6% and 11.0% of the applied radioactivity, respectively. MAI, MIT,

TMG and MIO were also identified, but their amounts did not exceed

10% of the applied radioactivity.

[Thiazolyl-14

C]TI-435 degraded to three major breakdown products, i.e.

TZMU, formamide (FA) and MIT. Maximum values of 39.7% (TZMU),

16.1% (FA) and 11.8% (MIT) were analysed. MAI and TMG were

identified as minor degradation products (maxima <10% of the applied

radioactivity).At the end of the study after 18 days, 0.8% of the applied

radioactivity was photo-mineralised to CO2 from [nitroimino-14

C]TI-435,

whereas 34.1% was mineralised from [thiazolyl-14

C]TI-435. Organic

volatile compounds were detected only in trace amounts (<0.1% of the

applied radioactivity).

5.3 Conclusion The degradation in the dark controls was negligible. Sterile conditions

and a pH-value of 7 were maintained throughout the test. Validity

criteria of the test can be considered as fulfilled.

Considering the rapid photolytic breakdown determined at a pH value

and a temperature typical for a natural environment, solar radiation will

significantly contribute to the degradation of the test substance in aquatic

test systems. It can also contribute to the elimination of residues of TI-

435 by means of mineralization of the thiazole ring.

5.3.1 Reliability 1

5.3.2 Deficiencies No



Date 2006/09/08

Materials and Methods The applicant’s version is acceptable and indicates no relevant discrepancies.

Results and discussion The applicant’s version is acceptable with minor restrictions.

Remark to Item 4.4.8:

Under this item the applicant provides the experimental half-life and the

corresponding environmental half-life under midsummer solar light conditions at

Phoenix/USA. The result of the calculation of the equivalent exposure time for

midday midsummer at 40°, is here not presented.

In the study the ratio of the photon flow density of the xenon-lamp to that of

sunlight of the midsummer scenario (40° latitude) was calculated to be 1.25.

Therefore, exposure to xenon light in the test apparatus for one hour was

equivalent to an exposure to midday midsummer sunlight conditions (40° latitude)

of 1.25 hours.

Conclusion The applicant's version is adopted.

Reliability 1

Acceptability acceptable

Remarks






COMMENTS FROM ...

Date Give date of comments submitted








Remarks



Table A7_1_1_1_2-1: Description of test solution and controls

Criteria Details

Purity of water Milli-Q-water (conductivity: 21 mΩ, total organic

carbon: 21 ppb)

Preparation of test chemical solution The test substances were dissolved in acetonitrile and

a purity of ≥ 98.1% (nitroimino-label) and 100%

(thiazolyl-label) was determined. Aliquots of 0.8 mL

(nitroimino-label) and 0.19 mL (thiazolyl-label) were

diluted in 200 mL buffer solution, sonicated, shaken

and transferred into the test vessels.

Buffer solution: 0.01 mol/L phosphate buffer, pH 7,

autoclaved before use.

Test concentrations (mg a.s./L) Initial concentration TI-435 (C0): 0.3 mg/L

Temperature ( C) 25°C ± 1°C

Preparation of actinometer solution A solution of 0.01 M uranyl nitrate and 0.05 M oxalic

acid per litre was prepared and sub-samples of 5.0 mL

were irradiated for 10 minutes. The remaining oxalic

acid was determined by titration with a 0.01 M

KMnO4 solution.

Controls Triplicate dark controls per radiolabel

Identity and concentration of co-solvent Acetonitrile

Content: 0.4% v/v ([nitroimino-14

C]TI-435),

0.1% v/v ([thiazolyl-2-14

C]TI-435)

Table A7_1_1_1_2-2: Description of test system

Criteria Details

Laboratory equipment Test vessels: 350 mL quartz glass vessels with trap

attachment for volatile organic products and CO2, or

with glass stopper

Radiometer: Radialux Light Measuring Instrument

(Atlas/Xenotest Co.) equipped with a global sensor

(λ = 300 to 800 nm), sensor: about 2.2 cm above the

sample platform

Test apparatus Heraeus ®Suntest cabinet equipped with a xenon

lamp (λ > 290 nm)

Properties of artificial light source:

Nature of light source Xenon lamp

Emission wavelenght spectrum 280 to 830 nm

Light intensity Prior to test: 1035 W/m²

End of test: 1018 W/m²

Filters UV glass filter cutting off wavelengths < 290 nm



Table A7_1_1_1_2-3: Recovery and distribution of the radioactivity after photolysis of [nitroimino-14

C]TI-435 and [thiazolyl-14

C]TI-435 in buffer solution (values given in % of

applied radioactivity)

Condi-

tion

Expos.

[hours]

CO2 TI-435 TZMU MAI MIT TMG MU MG MIO HMIO FA Not

identif.*

Total

[nitroimino-14

C]TI-435

irradiat. 0 n.s. 100.1 n.d. n.d. n.d. <0.5 n.d. n.d. n.d. n.d. - 1.7 102.1

1.5 n.s. 74.3 10.4 1.3 1.3 0.7 n.d. <0.5 n.d. 3.0 - 5.6 97.0

4 n.s. 27.2 24.3 3.9 4.2 1.7 0.9 3.0 4.2 13.6 - 10.4 93.4

24 n.s. 0.7 29.3 4.2 5.7 1.5 4.0 11.0 4.4 26.6 - 9.6 97.0

120 n.s. 0.6 24.4 0.9 6.2 1.4 7.7 20.4 4.2 18.8 - 10.8 95.4

264 0.4 <0.5 24.0 n.d. 7.1 1.6 9.2 29.5 2.1 9.4 - 12.0 95.6

432 0.8 <0.5 18.7 n.d. 4.4 1.0 11.0 34.7 2.4 7.1 - 10.5 91.0

dark 432 <0.1 93.8 1.2 <0.5 <0.5 <0.5 0.9 <0.5 n.d. n.d. - 1.0 98.4

[thiazolyl-14

C]TI-435

irradiat. 0 n.d. 95.0 n.d. n.d. n.d. 2.4 - - - - n.d. 4.6 102.0

1.5 1.0 75.8 10.2 3.1 3.3 0.9 - - - - n.d. 4.8 99.0

4 3.9 25.3 31.4 6.5 8.3 2.0 - - - - 5.3 15.5 98.0

24 10.8 0.5 39.7 8.7 11.8 3.0 - - - - 9.9 13.9 98.2

120 20.9 n.d. 31.5 2.4 7.1 2.9 - - - - 16.1 16.0 96.9

264 27.6 n.d. 31.7 n.d. 3.7 2.2 - - - - 16.0 14.0 95.4

432 34.1 n.d. 27.5 n.d. 1.6 1.6 - - - - 14.1 15.4 94.4

dark 432 0.3 104.1 n.d. n.d. n.d. n.d. - - - - n.d. 1.9 106.3

* no single compound exceeded 10% of the applied radioactivity

n.s. not sampled

n.d. not detected


MAI: 3-Methylamino-1H-imidazo[1,5-c]imidazole

MIT: 7-Methylamino-4H-imidazo[5,1-b][1,2,5]thiadiazin-4-one

TMG: N-(2-chlorothiazol-5-ylmethyl)-N’-methylguanidine

MU: Methylurea

MG: Methylguanidine

MIO: 2-Methylamino-2-imidazolin-5-one

HMIO: 4-Hydroxy-2-methylamino-2-imidazolin-5-one

FA: Formamide

Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 July 2005

RCC project no. 852224 Page 1 of 5


Ready biodegradability

1 REFERENCE

Officialuse only

1.1 Reference Bealing, D.J., Watson, S. (1999): TI-435: Assessment of ready biodegradability by measurement of carbon dioxide evolution. Covance, Harrogate, England; unpublished report no. 586/162-D2145



1.2.2 Companies with letter of access

None

1.2.3 Criteria for data protection



2.1 Guideline study Yes Directive 92/69/EEC, C.4-C

2.2 GLP Yes

2.3 Deviations Yes On day 20, the minimum temperature recorded was 18.5°C (guideline requirement: 22 ± 2°C), but this was not considered to have influenced the outcome of the study.


3.1 Test material TI-435 PAI

3.1.1 Lot/Batch number 30036608

3.1.2 Specification PAI

3.1.3 Purity

3.1.4 Further relevant properties

Solubility in water (20°C) : 327 mg/L (see Morrissey & Kramer, 2000a)

3.1.5 Composition of Product

Not applicable

3.1.6 TS inhibitory to micro-organisms

No

3.1.7 Specific chemical analysis

None

3.2 Reference substance

Yes Sodium benzoate

3.2.1 Initial concentration of reference substance

According to guideline


3.3.1 Inoculum / test species



3.3.3 Test conditions See Table A7_1_1_2_1-3





3.3.4 Method of preparation of test solution

The test substance was insufficiently soluble in water to permit dosing from a concentrated aqueous stock solution. The test substance was weighed onto PTFE discs and added, together with the weighing supports, to the test vessels and the toxicity control.

3.3.5 Initial TS concentration

51.9 - 52.0 mg TS/L corresponding to a nominal concentration of 15 mg DOC/L

3.3.6 Duration of test 28 days

3.3.7 Analytical parameter

CO2 evolution

3.3.8 Sampling 2, 4, 6, 8, 10, 15, 16, 19, 23, 28 days

3.3.9 Intermediates/ degradation products

Not identified

3.3.10 Nitrate/nitrite measurement

No

3.3.11 Controls Reference substance control without test substance; reference substance control with test substance (toxicity control).

3.3.12 Statistics None

4 RESULTS

4.1 Degradation of test substance

4.1.1 Graph See Figure A7_1_1_2_1-1

4.1.2 Degradation Percentage degradation at the end of incubation: Sodium benzoate: 82.5% theoretical CO2 yield (mean of duplicates) TI-435: 1.5% theoretical CO2 yield (mean of duplicates) Toxicity control: 77% theoretical CO2 yield (65% theoretical CO2 yield after 15 days)

4.1.3 Other observations None

4.1.4 Degradation of TS in abiotic control

No abiotic control

4.1.5 Degradation of reference substance

See Figure A7_1_1_2_1-1

4.1.6 Intermediates/ degradation products

Not determined


5.1 Materials and methods

Directive 92/69/EEC, C.4-C, CO2 Evolution Test (Modified Sturm Test) The pH was measured in the blank and reference vessels, but due to technical reasons not in the vessels containing TI-435.

5.2 Results and discussion

The blank-corrected CO2 yield of TI-435 after 28 days of incubation was only 1.5% (mean of two trials) of the theoretical maximum yield (165 mg CO2). Therefore, TI-435 cannot be classified as readily biodegradable.





The biodegradation of the reference substance sodium benzoate was clearly above 60% of the theoretical yield within 14 days and the 10-day period (10% in the first 4 days). TI-435 present in the toxicity control did not inhibit the microbial degradation of the reference substance.

5.3 Conclusion The biodegradation of the reference substance sodium benzoate was above 60% of the theoretical yield within 14 days and the differences in degradation between the replicates was by less than 20%. This demonstrates the validity of the test. Under the test conditions, TI-435 was not readily biodegradable. In the toxicity control, TI-435 did not inhibit the microbial degradation of the reference substance.

5.3.1 Reliability 1


Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the

comments and views submitted


Date 2005-08-17

Materials and Methods applicant’s version is acceptable.

Results and discussion applicant's version is adopted

Conclusion applicant's version is adopted

Reliability 1


Remarks none

COMMENTS FROM ...


Materials and Methods Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. Discuss if deviating from view of rapporteur member state





Remarks



Table A7_1_1_2_1-1: Inoculum / Test organism

Criteria Details

Nature Activated sludge

Species Not applicable

Strain Not applicable

Source Sewage treatment plant treating predominantly domestic sewage

Sampling site Burley Menston sewage treatment works (Yorkshire Water), England

Laboratory culture Not applicable

Method of cultivation Not applicable

Preparation of inoculum for exposure None

Pre-treatment None

Initial cell concentration Not applicable

Table A7_1_1_2_1-2: Test system

Criteria Details

Culturing apparatus Test vessels

Number of culture flasks/concentration 2

Aeration device CO2-free air

Measuring equipment Barium hydroxide titration

Table A7_1_1_2_1-3: Test conditions

Criteria Details

Composition of medium Synthetic mineral salts medium containing potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate dihydrate, ammonium chloride, calcium chloride dihydrate, magnesium sulphate heptahydrate, ferric chloride hexahydrate, hydrochloric acid.

Additional substrate No

Test temperature 20.8 to 22.6°C On day 20, the minimum concentration recorded was 18.5°C. This minor fluctuation to the required temperature of 22 ± 2°C is not considered to have influenced the outcome of the study.

pH Start: 7.5 (Blank), not determined for TI-435 due to the risk of the pH electrode becoming coated with the undissolved test substance. End: 7.4 (Blank), 7.4 (TI-435)

Aeration of dilution water Yes Air-flow: 50 to 100 mL/min

Suspended solids concentration 30 mg/L



Figure A7_1_1_2_1-1: Percentage biodegradation in the CO2 Evolution Test



Inherent biodegradability

JUSTIFICATION FOR NON-SUBMISSION OF DATA Officialuse only

Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ ] Limited exposure [ ] Other justification [ X ]

Detailed justification:

Undertaking of intended data submission [ ]

-




Date

Evaluation of applicant's justification

Conclusion

Remarks

COMMENTS FROM OTHER MEMBER STATE (specify)





Remarks



Section A7.1.1.2.3 Annex Point IIIA XII.2.1

Biodegradation in seawater


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ ] Limited exposure [ X ] Other justification [ ]



-




Date


Conclusion

Remarks






Remarks




Aerobic biodegradation




X


-




Date


Conclusion

Remarks






Remarks


Sumitomo Chemical Takeda Agro Co., Ltd. Document IIIA, Section A7

Biocidal active substance: Clothianidin

Page 1-12

PT 18 January 2014

Section IIIA 7.1.2.1.2

Annex point IIIA, XII.2.1

Anaerobic biodegradation

1 Reference Official use only

1.1 Reference (2013),


1.2.1 Data owner


2 Guidelines and Quality Assurance

2.1 Guideline study

2.2 GLP Yes

2.3 Deviations

X



Page 2-12

PT 18 January 2014

3 Materials and Methods

3.1 Test material (Test substance and reference standards)

Common Name: Clothianidin (TI-435) Chemical name: (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-

methyl-2-nitroguanidine

Molecular formula: C6H8ClN5O2S Molecular weight: 249.68

3.1.1 Lot/Batch number

3.1.2 Specification

3.1.3 Purity

X


X


3.1.6 Method of analysis Reverse Phase HPLC method Radiochemical purity by HPLC and TLC.

Detection: UV

1 TI-435 is another name for clothianidin.



Page 3-12

PT 18 January 2014

3.2 Reference substance TMG

3.3 Test system Characterisation at 0 DAT: Veal calf manure:

Pig manure:

Chicken manure:

X


3.4.1 Test system Application of [14C]-clothianidin to 3 manures; veal calf, pig and chicken. X

3.4.2 Temperature 20 ± 2°C under anaerobic conditions for veal calf and pig and aerobic conditions for chicken.

3.4.3 pH NA


3.4.5 Number of replicates

3.4.6 Sampling Veal calf, pig and chicken manure was sampled at 0, 7, 14, 30, 59, 120 and 181 days after treatment (DAT)

3.4.7 Analytical methods Reverse Phase HPLC method

Detection: UV

TLC method

X

3.5 Preliminary test



Page 4-12

PT 18 January 2014

4 Results and Discussion

4.1

5 Applicant's Summary and Conclusion

5.1 Materials and methods The rate of transformation and nature of transformation products of [14C]-clothianidin was studied at 20 ± 2°C under anaerobic conditions for veal calf and pig manure and aerobic conditions for chicken manure. Samples were taken at 0, 7, 14, 30, 59, 120 and 181 DAT. Control samples (treated with non-radiolabelled clothianidin) were incubated in parallel.

Veal calf and pig manures were conditioned for 21 days at 20 ± 2°C under anaerobic conditions. Dispensed and conditioned manure samples,

, were further conditioned in individual glass vessels for 4 days

(anaerobic incubations) or 19 days (aerobic incubations) prior to clothianidin application. Vessels were maintained at 20 ± 2°C in the dark and had a flow of moistened nitrogen (veal calf and pig manure) or air (chicken manure) through them. The veal calf and pig manure was checked to ensure it was anaerobic prior to clothianidin application.

Samples were removed for analysis

immediately after application of [14C]-clothianidin and at times up to 181 DAT.

Degradation rates of clothianidin and its main metabolite were

X



Page 5-12

PT 18 January 2014

determined.


X

X



Page 6-12

PT 18 January 2014

There were no individual metabolites, other than TMG, detected at > 6% AR in any manure sample analysed,

Clothianidin and TMG were identified by co-chromatography with reference standards by HPLC and TLC.

Degradation rates for clothianidin determined using SFO kinetics are summarised below:

Manure type DT-50 (days) Veal calf 31.5 Pig 13.4 Chicken 16.2

SFO was considered the best fit

X

X

X

5.2.1 DT50 Degradation rates for clothianidin determined using SFO kinetics: X



Page 7-12

PT 18 January 2014

Manure type DT50 (days) Veal calf 31.5 Pig 13.4 Chicken 16.2

X

5.3 Conclusion Clothianidin degraded in veal calf and pig manures incubated under anaerobic conditions and in chicken manure incubated under aerobic conditions with DT-50 values of 32, 13 and 16 days, respectively.

The main degradation product was identified as TMG.

There were no other degradation products present at > 6% AR.

X

5.3.1 Reliability 1

5.3.2 Deficiencies None reported


Evaluation by Rapporteur Member State

Date 2014/05/14



Page 8-12

PT 18 January 2014

Materials and Methods



Page 9-12

PT 18 January 2014

Results and discussion



Page 10-12

PT 18 January 2014

Conclusion

Reliability

Acceptability

Remarks

Comments from ...

Date



Conclusion

Reliability

Acceptability

Remarks



Page 11-12

PT 18 January 2014



Page 12-12

PT 18 January 2014

Sumitomo Chemical Takeda Agro Co., Ltd. Clothianidin August 2006







-




Date







Conclusion

Remarks






Remarks



Aerobic aquatic degradation study





-




Date


Conclusion

Remarks






Remarks


Sumitomo Chemical Takeda Agro Co., Ltd. TI-435 May 2005


Section A7.1.2/02 and

A7.1.2.2.2/02

Annex Point IIIA XII 2.1

Rate and route of degradation in aquatic systems

including identification of metabolites and degradation

products

Water/sediment degradation study

1 REFERENCE

Official

use only

1.1 Reference Reddemann, J. (2000): Anaerobic aquatic metabolism of the active

ingredient TI-435.

Bayer AG, 51368 Leverkusen, Germany; unpublished report no. MR-

497/00

x




letter of access

None


protection




US EPA Pesticide Assessment Guidelines, Subdivision N, Chemistry:

Environmental Fate §162-3 Anaerobic Aquatic Metabolism, 1982

2.2 GLP Yes

2.3 Deviations No x

3 METHOD

3.1 Test material itroimino-14

C] TI-435

3.1.1 Lot/Batch number 11553/1

3.1.2 Specification Specific radioactivity: 3.78 MBq/mg



3.1.3 Purity


properties

Solubility in water (20°C): 327 mg/L


3.2 Reference

substance




TZU: N-(2-chloro-5-thiazolylmethyl)-urea

TZMU: N-(2-chloro-5-thiazolylmethyl)-N’-methylurea

TMG: N-(2-chloro-5-thiazolylmethyl)-N’-methylguanidine

TZNG: N-(2-chloro-5-thiazolylmethyl)-N’-nitroguanidine

MNG: N-methyl-N’-nitroguanidine

3.3 Test system

3.3.1 Water/sediment

systems

Water and sediment were taken from a pond located on the Bayer

research farm in Howe, Indiana, USA. The pond has no history of

chemical treatment or known run-off contamination from chemical

treatments.

For water/sediment characteristics see Table A7_1_2_2_2/02-1.




A7.1.2.2.2/02




products


3.3.2 Test system

sampling

The test system was sampled on June 4, 1997 and the samples were

shipped to the test facility on June 9, 1997.

3.3.3 Test system

equilibration

Prior to the start of the test, water and sediment were separated by

decanting, and stones and plant debris were removed from the moist

sediment. Dry weight of the sediment was determined to be 29.5%. On

July 7, 1997, 42 mL of pond water were added to 224 g moist sediment

(= 66 g dry weight) resulting in a water:sediment ratio of 3:1. Sucrose

(2.5 g) was added to each test system to enhance microbial growth to

speed up the process of getting anaerobic conditions. Then the

incubation devices were purged with argon to replace oxygen, closed

and pre-incubated at 20 1°C in the dark for about 7 weeks. One week

before treatment, the redox potential had dropped to –235 to –255mV

and –58 to -115mV in water and sediment, respectively, and the oxygen

content in the water was 0-1% of saturation.

3.3.4 Test conditions Test vessels: 250 mL Erlenmeyer flasks to which an inert, gas-tight bag

was connected for sampling of volatiles.

Agitation: none;

Oxygen conditions: anaerobic;

Light conditions: dark;

Temperature: 20 ±1°C.

3.3.5 Rate of application Based on a total annual use rate of 300 g a.s./ha and assuming overspray

of a water of 2m depth, the concentration in the water phase is 15 µg

a.s./L. Accordingly, a nominal amount of 3.0 µg a.s. was applied to each

test system taking into account the total water volume of 200mL.

3.3.6 Preparation of

application solution [Nitroimino-

14C]TI-435 was dissolved in acetonitrile (32.12 g

a.s./mL).

3.3.7 Application 93.4 µL of the application solution was applied to the water surface of

each test system using an Eppendorf Comforpette. Argon was passed

through the headspace of the test system for 10 minutes and

subsequently the test system was slightly shaken without disturbing the

sediment phase to achieve homogeneous distribution of the test

substance in the water phase. The test systems then were placed in a box

with a continuous nitrogen flow (50 mL/min).

x

3.3.8 Duration of test 360 days

3.3.9 Sampling and

extractions

Duplicate vessels were dismantled and processed at 0, 1, 3, 7, 14, 30,

59, 90, 135, 182, 240 and 360 days after treatment.

Before dismantling the test systems, N2 was sucked through the test

system and the gas tight bag at a rate of 200 mL/min. for 30 minutes to

eluate potential volatile compounds from the system. The gaseous

stream was directed through a trapping system for CO2 and organic

volatiles.

Immediately after opening of the test vessels, redox potential, pH and

oxygen content were measured in the supernatant water and the redox

potential was measured in the sediment.

At each sampling date, the supernatant water was carefully separated

from the sediment. An aliquot was used for determination of radioactive

CO2 and/or carbonates. The remaining water was centrifuged, the

precipitate added to the sediment phase, and an aliquot of the

supernatant was concentrated under reduced pressure and subjected to

x




A7.1.2.2.2/02




products


TLC analysis.

The sediment first of all was extracted using organic solvents, i.e. three

times with acetonitrile, then once with acetonitrile/H2O (50:50, v/v),

followed by one extraction with dichloromethane. The extracts were

combined, concentrated and subjected to TLC. In addition, aliquots of

sediment were extracted with methanol : water (1 : 1, v/v) under reflux

for 2 h (all samples), with 0.05M 1-pentane-sulfinic acid in methanol :

water (7 : 3, v/v) under reflux for 2h (from day 1 onwards) or with

acetonitrile : 1M HCl (7 : 3, v/v) under reflux for 4 h (from day 3

onwards). In regards of the extracted radioactivity, only the extracts

from the acetonitrile-HCl extraction procedure were analysed by TLC.

The amount of radioactivity remaining in the extracted sediment (“non

extractable”) was determined by combustion.

3.3.10 Controls Two test systems without and two systems with test substance treatment

were set up in addition for determination of the biological activity in the

sediment in the course of the test period


Concentration of test substance and degradation products: TLC on Silica

gel 60 plates.

Solvent system:

i) dichloromethane/acetonitrile/acetic acid 70/30/1 (v/v/v)

ii) butanol/water/acetone/acetic acid 65/10/5/15 (v/v/v/v)

Solvent system i) was used for water phase and all extracts, whilst

solvent system ii) was used additionally for a second development of the

TLC plates with the acetonitrile/1M HCl extracts for separation of polar

components.

LOQ for a single component in the water and organic phase extracts was

1% of the applied radioactivity, and 2% for the acetonitrile/1M HCl

extracts.

3.3.12 Intermediates/

degradation

products

No degradate was formed at amounts >4.5% of the applied radioactivity,

Parent TO-435 was confirmed by TLC, HPLC-MS.

3.3.13 Calculation of

dissipation rates

Dissipation of TI-435 from the water phase, the sediment and the whole

test system was calculated using the computer software ModelManager

Vers. 1.1.

4 RESULTS

4.1 Test conditions

during incubation

Anaerobic conditions were demonstrated by the measurements of redox

potential and dissolved oxygen. During the test period, redox potential

varied between –113 and –212mV and between –123 and –210mV in

the water and sediment phase, respectively, and the oxygen content in

the water phase did not exceed 2% of maximum saturation at any

sampling date.

pH values ranged from 4.7 to 5.0 between days 0 and 135, and increased

thereafter to pH 6.7 to 7.1.

The mean temperature was 20.29°C (min/max: 19.90/20.90°C).

4.2 Recovery Three aliquots of the application solution were measured by LSC during

the application procedure and the mean (3.09 g a.s./test system) was

taken as the initial 100% value.




A7.1.2.2.2/02




products


Within the course of the study, the total recoveries varied between

95.1% and 101.7% of the applied radioactivity (for details see Table

A7_1_2_2_2/02-2).

4.3 Distribution of

radioactivity

During the course of the experiment, the amount of radioactivity in the

water phase decreased from 89.8% of applied on day 0 to 1.4% on day

360, whereas the radioactivity in the sediment increased from 9.9% to

95.2% in the same period. The amount of extractable residues was 9.4%

on day 0, increased to a maximum of 46.4% on day 14 and declined to

14.3% by the final sampling. Bound residues increased from 0.5% on

day 0 to a maximum of 82.6% on day 182, and decreased thereafter to

80.9% on day 360.

For details see Table A7_1_2_2_2/02-2.

x

4.4 Identification of

radioactivity

4.4.1 Degradation of the

test substance

In the water phase, the amount of TI-435 steadily decreased from 87.4%

of applied radioactivity on day 0 to below the limit of detection, i.e.

<1% of applied, on day 135.

In the sediment, the amount of TI-435 increased from 9.0% on day 0 to

a maximum amount of 41.2% of the applied radioactivity on day 3 and

decreased thereafter to below the limit of detection on day 240.


x

4.4.2 Formation of

degradation

products

In the water phase, only the parent compound was found in quantifiable

amounts. No additional fraction was detected by TLC at an amount

above the LOD of 1% of applied radioactivity.

In the sediment, five polar fractions were detected none of them ever

exceeding an amount of 4.3% of the applied radioactivity. The retention

time of any of those fractions did not match with any of the available

reference substances, and additional identification efforts were not

feasible in regards of the low amounts of the individual fractions.


4.4.3 Mineralisation &

volatile organic

compounds

Neither CO2 nor any organic volatile compound was detected in the test

systems at significant amounts at any sampling date. Recovery was

always < 0.1% of the applied radioactivity


4.5 Half-life of the test

substance

The half-life of TI-435 in the water phase, sediment and the whole

system is presented in Table A7_1_2_2_2/02-3.

x


5.1 Materials and

methods

US EPA Pesticide Assessment Guidelines, Subdivision N, Chemistry:

Environmental Fate §162-3 Anaerobic Aquatic Metabolism, 1982.

The degradation of [nitroimino-14

C]TI-435 was investigated in one US

water/sediment system (Howe farm pond). The test substance was

applied at a concentration of 15 µg a.s./L water (based on the use rate of

300 g a.s./ha and overspray of a water of 2 m depth) to the aquatic

system consisting of 66 g sediment (dry weight) and 200 g water. The

test systems were incubated in the dark at 20°C for 360 days and 0, 1, 3,

7, 14, 30, 59, 135, 182, 240 and 360 days after treatment, duplicate

vessels were processed.




A7.1.2.2.2/02




products


5.2 Results and

discussion

The amount of radioactivity detected in the water phase rapidly

decreased within the course of the study to levels of 1.4% of the

applied radioactivity on day 360. Bound residues in the sediment

reached on average 81% at the end of incubation.

No degradates were found in the water phase and five not identified

polar fractions occurred in the sediment, none of them exceeding a

maximum amount of 4.3% of the applied radioactivity. Volatiles

including CO2 accounted for less than 0.1% of the applied radioactivity

at any sampling.

DT50 of TI-435 was calculated to be 4, 11 and 21 days for the water

phase, sediment phase and the whole system, respectively.

Total recoveries of the applied radioactivity ranged from 95.1% to

101,7%. Validity criteria of the test can be regarded as fulfilled.

5.3 Conclusion Under anaerobic conditions, TI-435 disappears rapidly from the water

phase to the sediment, with non-extractable residues forming the major

sink in time.

5.3.1 Reliability 1





Use separate "evaluation boxes" to provide transparency as to the



Date 2007-01-10

Materials and Methods Apart from smaller discrepancies (see below), applicants version is acceptable.

Comments:

2.3. Deviations: Yes; deviating to the study protocol, the results of the sterile

samples were not reported since no degradation of the parent compound was

observed and no further information relevant for the study was indicated.

More specifically:

1.1.Reference includes data from Amendment no.: 1 to “Anaerobic aquatic

metabolism of the active ingredient TI-435” by Reddemann, J. (2001).

Bayer AG, 51368 Leverkusen, Germany; unpublished report no. MR-497/00

3.3.7. Application: after each application the aqueous phase was gently mixed by

rotating the incubation vessels…

3.3.9. Sampling and extractions: the extracted sediment was stored up to one year

at about –20°C until an aggravated extraction was performed. Stability studies

showed that TI-435 was stable under the aggravated extraction conditions applied.

Because of the low extracted amount of radioactivity in case of exhaustive and

hot extraction, only the acid hot extracts were analyzed by means of TLC within a

few days after preparation. (amendment to last two paragraphs)

Results and discussion Despite minor reporting deficiencies (see below) the applicant´s version is

adopted.

More specifically:

4.3: Distribution of radioactivity: Bound residues……slightly decreased

thereafter….(amendment to last sentence)

4.4.1 Degradation of the test substance: In the entire system the amount of TI-435

gradually declined from 96.4% of applied radioactivity at day 0 to < 1% at 240

days post treatment.

4.5 Half-life of the test substance: The DT-values presented in the Table are

according to non-linear first order kinetics.

Conclusion The applicant´s version is accepted.

The partitioning and degradation behaviour (fate) of clothianidin was studied in

an anaerobic aquatic system using pond water and corresponding sediment.

During a period of 360 days, decrease of clothianidin in the water phase is rapid

with a calc. non-linear first-order dissipation half-life of 4 days; the corresponding

half-life for the entire system being 21 days. Concurrently, in the sediment the

amount of non-extractable residues steadily increased to reach 81% after 360

days. Metabolism of clothianidin in the entire system is insignificant (no

metabolite > 5% was found). Ultimate biodegradation is negligible (< 0.1% CO2

at any sampling date).

The results of the anaerobic water/sediment study show a rapid translocation of

clothianidin from the water phase to the sediment with resultant formation of a

very high plateau of bound residues, both negligible metabolism and

mineralization.

Reliability 1


Remarks none

COMMENTS FROM ... (specify)











Remarks



System Pond on Howe Farm, IN, USA

Supernatant water

Total organic carbon [mg/L]

Dissolved organic carbon [mg/L]

Total water hardness [°dH]

9.7

9.7

2.5

Sediment

Particle size:

sand [%]

silt [%]

clay [%]

29.1

50.2

20.7

Sediment type (according to USDA) silt loam

Organic carbon [mg C/100 g dry mass] 6.21

Nitrogen [mg N/100 g dry mass] 540

Phosphorus [mg P/100 g dry mass] 981

pH (aq. dest.)

(CaCl2)

5.8

5.0

Cation Exchange Cap. [meq/100 g dry mass] 28

Respiratory activity of microflora

Day 0 [mg CO2/h/kg dry sediment]

Day 360 [mg CO2/h/kg dry sediment]

10

8

Table A7_1_2_2_2/02-2: Degradation of TI-435 and formation of metabolites in water/sediment systems

(values are given as % of applied radioactivity)

Days after Pond on Howe Farm, IN, USA

application 0 1 3 7 14 30 59 90 135 182 240 360

WATER PHASE 89.8 81.7 53.4 41.2 22.6 11.9 4.0 2.5 1.1 0.6 0.3 1.4

Origin 1.0 1.3 2.0 n.d. n.d. 1.1 1.5 1.6 1.1 n.d. n.d. 1.3

TI-435 87.4 80.0 50.7 40.1 21.4 10.9 2.7 1.0 n.d. n.d. n.d. n.d.

diffuse radioact. 1.4 n.d. 1.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

SEDIMENT* 9.9 20.0 47.0 57.3 75.8 84.2 91.2 92.6 96.6 95.9 95.3 95.2

extractable 9.4 18.0 42.1 44.6 46.4 37.6 24.8 11.7 16.4 13.3 18.2 14.3

Origin n.d. n.d. n.d. n.d. 3.7 4.1 6.5 2.4 7.8 6.5 6.5 13.2

TI-435 9.0 17.8 41.2 40.6 38.0 22.5 6.7 2.4 2.3 1.3 n.d. n.d.

PZ1-5** n.d. n.d. n.d. 2.0 3.0 8.6 6.9 2.3 6.5 5.3 8.6 n.d.

diffuse radioact. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

not extractable 0.5 2.0 4.9 12.9 29.3 46.6 66.4 80.9 80.1 82.6 77.0 80.9

14CO2 - <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

TOTAL 99.7 101.7 100.4 98.6 98.3 96.1 95.2 95.1 97.7 96.5 95.6 96.6

n.d. : not detected (< 1% of the applied radioactivity; < 2% of the applied radioactivity for hot acid extraction)

* comprises findings of extractions with organic solvents and hot acid (acetonitrile : 1M HCl, 7 : 3)

** sum of 5 not identified polar metabolites (amount of individual metabolites varied between 2.0 and 4.3%

of the applied radioactivity)



Table A7_1_2_2_2/02-3: Dissipation times of TI-435 in an aquatic system

Compartment DT50 DT90

Water 4.0 days 37.8 days

Sediment 11.2 days 37.2 days

Entire system 21.0 days 63.0 days



Section A7.1.3 Annex Point IIA7.7

Adsorption/desorption screening test

1 REFERENCE

Official use only

1.1 Reference Lewis, C.J. (2000): [14

C]TI-435: Adsorption/desorption in soil.

Covance, Harrogate, England; unpublished report no. 586/139-D2142




letter of access

None


protection




OECD 106 (May 1981), US EPA Subdivision N, Section 163-1 and

Environmental Chemistry and Fate Guideline for Registration of

Pesticides in Canada Section B (1987)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material [thiazolyl-14

C]TI-435

3.1.1 Lot/Batch number A-980501


968.76 MBq/mmol

3.1.3 Purity Radiochemical purity: >99%

Chemical purity: 98.3%


properties

Solubility in water (20°C) : 327 mg/L


3.1.5 Method of analysis Identification of TI-435 and reference substances:

HPLC or TLC.

Measurement of radioactivity: LSC

3.2 Degradation

products

Degradation products tested: Yes

At any time of the test all degradation products accounted for < 1% of

the a.s. added.


for degradation

products

See 3.1.5

3.3 Reference

substances

Yes (for identification of parent compound and

possible degradation products):



CTNU: N-(2-chlorothiazol-5-ylmethyl)-N’-





nitrourea

TZMU: N-(2-chlorothiazol-5-ylmethyl)-N’-

methylurea

ACT.HCl: 2-chlorothiazol-5-ylmethylamine hydrochloride


for reference

substance

See 3.1.5

3.4 Soil types Available data are given in Table A7_1_3-1


3.5.1 Test system Adsorption and desorption of TI-435 was

measured using a batch equilibrium procedure to

determine Koc values of [thiazolyl-14

C]TI-435 in

three US and two European soils.

3.5.2 Test solution and

Test conditions

The test substance TI-435 was tested in a concentration range of 0.04 to

5.0 µg a.s./mL.

3.6 Test performance

3.6.1 Preliminary test According to OECD 106

Degree of saturation: 5 µg a.s./mL

Equilibration: 49 hours for the adsorption and

24 hours for the desorption

Soil/solution ratio: 5:1 w/w, 2:1 w/w and 1:1 w/w

3.6.2 Screening test:

Adsorption

According to OECD 106


Desorption

According to OECD 106

3.6.5 Other test None

4 RESULTS

4.1 Preliminary test The obtained solution and equilibration time is

acceptable; the applicability of the test substance

TI-435 is given.

4.2 Screening test:

Adsorption

Solid weight: 10 g

Supernatant volume: 10 mL

Degree of adsorption: 24-42%, 74-90%, 47-70%,

53-76% and 28-43% for Quincy, Elder, Crosby,

Laacher Hof and BBA 2.1 soil, respectively.

4.3 Screening test:

Desporption

Solid weight: 10 g

Supernatant volume: 10 mL

Degree of desorption: 24-41%, 6-15%, 17-27%, 14-26% and 31-37% of





adsorbed TI-435 for Quincy, Elder, Crosby, Laacher Hof and BBA 2.1

soil, respectively.

4.4 Calculations

4.3.1 Ka, Kd Calculations of adsorption and desorption constants are given in Table

A7_1_3-2

4.3.2 Kaoc, Kdoc Calculations of adsorption and desorption constants are given in Table

A7_1_3-2

4.5 Degradation

products

TI-435 was found to be stable (< 1% degradation) during both

processes, i.e. adsorption and desorption.


5.1 Materials and

methods

The test system is described in 3.5.1 (batch equilibrium procedure). The

OECD guideline is fulfilled, no relevant deviations from the guideline

occurred.

5.2 Results and

discussion

The Kaoc –values varied between 84 and 345 and

were in a tighter range than the Freundlich

isotherm (0.52 to 4.14). This indicates that the

organic carbon content of the soil is of great

importance for the adsorption of TI-435. The

Kdoc–values (95 to 382) were higher than the

adsorption constants for all soils indicating a

partial irreversible adsorption.

There was a good correlation between the concentrations adsorbed and

in solution for the concentration range tested.

5.2.1 Adsorbed a.s. [%] The percentage adsorption of test substance varied between 24 and 90%

of the applied a.s. depending on soil type and concentration.

5.2.2 Ka 0.52 - 4.14 mg/g

5.2.3 Kd 0.62 - 4.58 mg/g

5.2.4 Kaoc 84 - 345 mg/g (mean: 160 mg/g) x

5.2.5 Ka/Kd 0.69 - 0.90 (mean: 0.84)

5.3 Conclusion Based on the classification of MCCALL ET AL. (1980), TI-435 is

classified as being medium to highly mobile in the soils tested.

5.3.1 Reliability 1




Date 2006-05-19

Materials and Methods The applicant’s version is adopted.




Results and discussion The applicant’s version is adopted.

Comment to Items 5.2.2; 5.2.3.; 5.2.4:

For the adsorption/desorption coefficients Ka and Kd as well as for the Kaoc value

the wrong units are used. The units have to be changed from mg/g to ml/g.

Conclusion Chlothianidin is classified as being medium to highly mobile in the soils tested.

The geometric mean value of the KaOC (applied in the environmental exposure

assessment) amounts to 140 ml g-1

.

Reliability 1


Remarks

COMMENTS FROM

Date Give date of the comments submitted








Remarks



Table A7_1_3-1: Classification and physico-chemical properties of soils used as adsorbents

Quincy Elder Crosby Laacher Hof BBA 2.1

Origin Grant County,

WA, USA

Watsonville,

CA, USA

New Holland,

OH, USA

Monheim,

Germany

Jockgrim,

Germany

Sand (2000 - 53 µm) 76 59 27 70 93

Silt (53 – 2 µm) 20 25 43 16 3

Clay (< 2 µm) 4 16 30 14 4

Classification (acc. to USDA) loamy sand sandy loam clay loam sandy loam sand

organic carbon [%] 0.4 1.2 1.2 2.1 0.5

organic matter [%] 0

.

7

2

.

1

2

.

1

3

.

6

0

.

9

pH

(water)

8

.

4

7

.

6

6

.

6

6

.

8

4

.

1

pH (1

M KCl)

7

.

2

6

.

2

5

.

2

6

.

2

3

.

7

CEC

[mEq/1

00 g]

3

.

0

1

2

.

7

6

.

8

7

.

4

3

.

3

WHC

(at pF

0)

3

3

.

7

4

2

.

9

5

0

.

1

5

4

.

8

3

2

.

4

CEC = cation exchange capacity WHC = water holding capacity



Table A7_1_3-2: Adsorption and desorption constants of [14

C]TI-435 in five different soils

Soil Adsorption Desorption Mobility*

Ka Kaoc 1/n Kd Kdoc 1/n Kddes (serial)**

Quincy 0.52 129 0.8351 0.62 154 0.8068 0.69 high

Elder 4.14 345 0.8088 4.58 382 0.8115 3.88 medium

Crosby 1.48 123 0.8216 1.67 139 0.8240 1.66 high

Laacher Hof 1.77 84 0.8146 1.99 95 0.8136 1.98 high

BBA 2.1 0.59 119 0.8648 0.85 170 0.8843 0.90 high

MEAN n.a. 160 0.8290 n.a. 188 0.8280 n.a.

* classification according to MCCALL ET AL. (1980)

** serial desorption at 5 µg/mL according to the Canadian guideline

n.a. not applicable


Section A7.1.4.1 Annex Point IIIA XII.2.1

Field study on accumulation in the sediment


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ X ] Limited exposure [ ] Other justification [ ]



-




Date


Conclusion

Remarks






Remarks



Section A7.1.4 Annex Point IIIA XII.2.2

Further studies on adsorption and desorption in water/sediment systems




X


-




Date


Conclusion

Remarks






Remarks





A7.2.2.1/02 Annex Point IIIA XII 1.1

Aerobic degradation in soil, initial study

The rate and route of degradation including the

identification of the processes involved and identification

of any metabolites and degradation products in at least

three soil types under appropriate conditions

1 REFERENCE

Official

use only

1.1 Reference Schad, T. (2000c): Aerobic degradation and metabolism of TI-435 in six

soils.


419/99

x




letter of access

None


protection




SETAC (1995) and US EPA Subdivision N, 162-1

2.2 GLP Yes

2.3 Deviations No



C]TI-435

3.1.1 Radiolabelling [thiazolyl-2-14

C]TI-435


3.1.3 Specific

radioactivity

3.84 MBq/mg

3.1.4 Purity Radiochemical purity: > 99% according to both radio HPLC and TLC


3.1.5 TS inhibitory to

microorganisms

No

3.2 Reference

substance







3.3 Test system

3.3.1 Soils The route and rate of degradation was investigated in 6 US soils

For soil characteristics see Table A7_2_1_02-1.










3.3.2 Test system

sampling

All soils were taken freshly from US fields and shipped immediately to

the test facility. Prior to the start of the test, they were stored for a few

days in a refrigerator to maintain their biological activity.

3.3.3 Test system

preparation

A few days before starting the test, the soils were carefully air dried and

sieved to a particle size of 2 mm. Subsequently, the soil moisture

capacity was determined. The microbial biomass was determined

immediately before the start and within the course of the study

according to ANDERSON AND DOMSCH (1978).

3.3.4 Test conditions Incubation system: 300 mL Erlenmeyer flasks closed with a trapping

attachment (containing soda lime and quartz wool) that permitted some

gas exchange but which adsorbed potential 14

C-volatiles including CO2;

Oxygen conditions: aerobic;


Temperature: 20 ± 1°C;

Soil moisture: 75% of the 1/3 bar moisture content for all soils.

3.3.5 Rate of application 13.3 µg a.s./100 g dry soil, equivalent to an annual rate of

approximately 300 g a.s./ha (assuming 15 cm soil depth and 1.5 g/cm3

soil density).

3.3.6 Preparation of test

solution and

application

An adequate amount of the test substance (dissolved in acetonitrile) was

applied to an aliquot of about 40 g soil, which was then homogeneously

mixed into the remaining total amount of soil using a tumble mixer.

Aliquots corresponding to 100 g of dry soil were weighed into the test

vessels (for all soils except Crosby only 50g treated soil were incubated

in the test vessel for the sampling day 181).

3.3.7 Control of moisture

content

The test vessels were weighed monthly. A loss of soil moisture was

balanced by the addition of appropriate amounts of distilled water. Prior

to opening the incubation vessel, volatile compounds possibly still

present in the headspace of the vessel were transferred into the trapping

attachment by purging with water-saturated air.

3.3.8 Duration of test 181 days for all soils except Crosby for which an additional sampling

was done after 379 days.

x

3.3.9 Sampling and

extractions

Duplicate or single vessels were processed on 0 (>1 hour), 1, 7, 62, 120

and 181 days after treatment. In case of soil Crosby duplicate test

systems were additionally sampled on day 379 after treatment.

At each sampling date, the soil was extracted four times with

acetonitrile followed by one extraction step with water. Then residual

radioactivity in soil was determined via LSC following combustion of

soil aliquots. To obtain some information on the nature of residues

remaining in soil, the soils were additionally subjected to hot extraction

with acetonitrile : water (50:50, v/v) by refluxing for 2 hours.

x

3.3.10 Biomass

determination

For the purpose of biomass determination at the beginning and in the

course of the study period (days 0 and 181 for all soils, and in addition

on day 379 for soil Crosby), samples with and without active ingredient

were incubated under identical conditions as in the main experiment.

3.3.11 Analytical methods Radioactivity: liquid scintillation counting (LSC); directly (for liquid

samples e.g. extracts) or after combustion (for soil samples only)

Concentration of test substance and degradation products: TLC on










a) Silica gel RP-18 F-254 plates; mobile phase: methanol/acetone/water

(10:10:80, v/v/v); vertical development;

b) Silica gel NH2 F-254 plates; mobile phase: i) methanol for 3 minutes

followed by ii) acetonitrile/Merck Titrisol buffer pH2 (8.8:0.2, v/v) for

15 minutes; horizontal development.


degradation

products

Identified

Degradation products were identified by TLC co-chromatography and

detection in the UV chamber at 254 nm. Radioactive regions were

quantified by the software package Tina (version 2.08, Raytest).

4 RESULTS

4.1 Recovery The actually applied radioactivity was determined by combustion of

small aliquots of each soil immediately after homogenisation. These

values were taken as 100% for all further data evaluations.

The radioactivity determined on day 0 ranged between 96.4% and

103.6% of the applied radioactivity. During the course of the study, the

total recoveries varied between 87.1 and 96.4% of the applied

radioactivity in soil Crosby, 99.0 and 101.6% in soil Elder, 96.8 and

105.0% in soil Fuguay, 101.9 and 105.0% in soil Quincy, 99.1 and

103.1% in soil Sparta and between 96.5 and 99.8% in soil Susan.

4.2 Extracted and

non-extracted

radioactivity

With the exception of soil Fuguay, the amount of extractable residues

decreased with time primarily due to the increase in bound residues and

mineralisation.

The most significant decrease in extractable radioactivity occurred in

soil Crosby, i.e. from 94.0% on day 0 to 68.8% on day 181 and 65.1%

on day 379. In the other soils, extractable radioactivity decreased from

95.8 to 101.9% on day 0 to 81.4 to 91.2% on day 181. The non-

extractable radioactivity in all soils varied between 3.7 and 11.7% of

applied radioactivity on day 181 of incubation, and was 9.5% in soil

Crosby after 379 days.

For details see Table A7_2_1_02-2.

4.3 Degradation of the

test substance

After 181 days, between 63.6 and 95.3% of the applied radioactivity

represented TI-435. In soil Crosby, which was incubated for 379 days,

the amount of TI-435 decreased to 60.3% at the end of the test period.


4.4 Mineralisation After 120 days of incubation, 1.5 to 8.1 of the applied radioactivity was

mineralised to 14

CO2 in the six soils, and the release of 14

CO2 increased

further to values between 2.1 and 10.7% after 181 days. After 379 days

incubation of the Crosby soil, 16.9% of the applied radioactivity was

detected as 14

CO2. This soil showed the highest mineralisation rate up

from sampling day 62.

No additional volatile products were detected (<0.1% of applied

radioactivity).










4.5 Metabolites and

degradation

products

TZNG and TZMU were the only metabolites identified in all soils.

Maximum amounts in the different soils varied between 0.2 and 0.7% of

applied radioactivity for TZNG and between 0.3 and 1.8% of applied

radioactivity for TZMU. Unidentified radioactivity can be differentiated

in radioactivity remaining at the origin of TLC and diffuse radioactivity

spread over the TLC lane, with maximum amounts varying between 1.3

and 3.9% and 1.5 and 2.0% of applied radioactivity, respectively.


4.6 Degradation rate

x

4.7 Degradation route The proposed degradation pathway of TI-435 in soil is presented in


5.1 Materials and

methods

Aerobic degradation and metabolism of TI-435 was investigated in six

US soils according to SETAC (1995) and US EPA Subdivision N, 162-1.

No deviations occurred. The test substance was applied at a

concentration of 13.3 µg a.s./100 g dry soil, equivalent to an annual

application rate of approximately 300 g a.s./ha assuming 15cm soil depth

and bulk density of 1.5. The soils were incubated in the dark at 20°C for

181 days, with the exception of soil Crosby that was incubated for 379

days.

5.2 Results and

discussion

DT50 values for the degradation of TI-435 in soil ranged between 533

and 1328 days. However, these values have to be treated very carefully

since they were extrapolated far beyond the experimental period.

5.3 Conclusion Validity criteria can be considered as fulfilled.

5.3.1 Reliability 1















Date 2007-01-10

Materials and Methods Apart from some amendments (see below) the applicants version is acceptable.

More specifically:

1.1 reference: included are data from amendment No.: 1 by Schad, T. (2001):

Aerobic degradation and metabolism of TI-435 in six soils.

Bayer AG, 51368 Leverkusen, Germany; unpublished report no. MR-419/99

3.3.8: Duration of test: the metabolism part of the study was performed with soil

Crosby and therefore the test duration was 379 days in contrast to the examination

of the degradation kinetics, which was performed in the other five soils with a test

duration of 181 days.

3.3.9 Sampling and extractions: the amount of bound residues was calculated as

remaining radioactivity in soil (after first extraction at room temperature)

substracted by the radioactivity measured in the second extracts. The radioactivity

measured in the filters (< 3,7%) were added to bound residues. Hence, the

radioactivity which was not extracted by the first and second extraction procedure

was regarded as bound residue.

Results and discussion Apart from some reporting deficiencies (see below) the applicants version is

acceptable.

In the six aerobic laboratory soil degradation tests, the amount of extracted

radioactivity only slightly decreased with time with up to 95,2 % of unchanged

clothianidin still identified after 120 days. First order DegT 50 values determined

for clothianidin in the individual soils amounted to more than one year . Two

metabolites, TZNG and TZMU, were detected in the soil extracts, none of which

exceeding 1,8% of applied radioactivity. Unidentified radioactivity amounted to a

maximum amount of 3,9%. Mineralization was negligible accounting for a max. of

8,1 % after 120 days resp. max. 16,9 % in one soil after 379 days. The amount of

bound residues slightly increased towards the end of the study with a max. of 9.9%

after 120 days resp. 11.7% in one of the six soils after181 days.

More specifically:

4.6 Degradation rate: calculations were performed using the computer software

Microsoft Excel 97, the results rounded to one or two digits. The mathematical

assessment of the metabolism data was performed using the ASCL Optimise

Software package (MGA Software, 1996). All reaction steps were assumed to be

of first order. The corresponding correlation coefficients (R2) for the DT 50 values

calculated varied between 0,7176 (Susan) and 0,9987 (Quincy) with 0,9595 for

Elder. In soil Fuguay > 95% total radioactivity were still identified after 181 days.

Conclusion The applicant's summary and conclusion is acceptable. In light of DT 50 values up

to more than one year due to both negligible metabolism and ultimate

biodegradation, clothianidin is in an overall assessment highly persistent in soil

Reliability 1


Remarks none










COMMENTS FROM ...









Remarks



Soil Crosby Elder Fuguay Quincy Sparta Susan

Soil type (according to USDA) silt loam loam loamy sand loamy sand sand silt loam

Particle size (according to USDA)

sand 2000-50 µm [%]

silt 50-2 µm [%]

clay < 2 µm [%]

17.7

58.8

23.5

50.2

38.1

11.7

77.2

19.8

3.0

79.6

13.6

6.8

92.1

7.0

0.9

18.7

53.9

27.4

Particle size (according to DIN)

sand 2000-63 µm [%]

silt 63-2 µm [%]

clay < 2 µm [%]

15.6

60.9

23.5

47.0

41.3

11.7

74.4

22.6

3.0

-

-

-

92.0

7.1

0.9

14.3

58.3

27.4

pH (water)

(CaCl2)

6.74

6.01

6.67

5.84

6.67

5.84

6.8

-

6.22

5.31

6.66

5.91

organic carbon [%]

organic matter [%]

1.37

2.36

1.41

2.43

0.35

0.6

0.4

0.8

0.73

1.26

3.27

5.62

CEC [meq/100 g soil] 15 18 5 6.08 6 30

Microbial biomass*

Day 0 [mg microbial C/kg soil]



476

244

177

195

135

n.d.

16

25

n.d.

176

54

n.d.

116

25

n.d.

498

409

n.d.

75% of 333 mbar moisture

[g water to 100 g dry soil]

19.85 16.91 9.6 12.65 5.36 30.58

* On day 0 determined in soil without active substance. On day 181 and 379 determined in soil containing the active substance.

n.d.: not determined



Soil Days

after

appli-

cation

14CO2

Not

extracted

radio-

activity

Extracted

radio-

activity

Origin

TI-435

TZNG

TZMU

Diffuse

radio-

activity

Total

Crosby 0 n.m. 2.5 94.0 0.2 93.7 n.d. n.d. 0.1 96.4

silt loam 7 0.1 2.5 89.9 0.2 89.6 <0.1 <0.1 <0.1 92.4

62 5.3 4.7 83.2 1.1 80.2 0.2 0.8 0.9 93.2

120 8.1 3.5 80.7 1.2 76.2 0.7 1.6 0.9 92.4

181 10.7 7.7 68.8 1.6 63.6 0.7 1.4 1.5 87.1

379 16.9 9.5 65.1 1.7 60.3 0.5 1.4 1.3 91.4

Elder 0 n.m. 2.1 97.7 0.2 97.5 n.d. n.d. 0.1 99.8

loam 7 0.2 2.8 97.2 0.1 97.1 n.d. n.d. n.d. 100.1

62 1.2 1.5 99.0 n.d. 98.6 n.d. n.d. 0.4 101.6

120 2.0 1.9 97.3 1.1 95.2 0.1 0.3 0.6 101.2

181 2.5 5.3 91.2 1.5 87.5 0.2 0.3 1.8 99.0

Fuguay 0 n.m. 1.7 95.4 0.1 95.3 n.d. n.d. <0.1 97.1

loamy 7 0.2 3.2 98.8 0.7 98.0 n.d. n.d. n.d. 102.2

sand 62 0.8 2.0 98.8 0.3 97.9 n.d. 0.2 0.4 101.7

120 1.5 2.6 92.7 2.0 89.3 n.d. 0.6 0.8 96.8

181 2.1 3.7 99.2 1.4 95.3 0.1 0.8 1.8 105.0

Quincy 0 n.m. 1.7 101.9 <0.1 101.9 n.d. n.d. <0.1 103.6

loamy 7 0.8 2.4 100.2 0.8 99.3 <0.1 n.d. n.d. 103.3

sand 62 4.0 4.3 96.7 1.2 93.1 0.2 1.1 1.1 105.0

120 5.6 6.4 90.0 1.8 85.8 0.4 1.5 0.6 101.9

181 7.0 8.3 86.9 1.9 80.8 0.5 1.8 2.0 102.2

Sparta 0 n.m. 2.5 100.6 0.1 98.9 n.d. n.d. 1.6 103.0

sand 7 0.6 2.9 98.7 0.1 98.6 n.d. n.d. n.d. 102.2

62 3.3 4.4 95.3 1.1 93.5 n.d. 0.2 0.5 103.0

120 4.8 9.9 88.3 3.5 82.5 0.3 0.6 1.4 103.1

181 5.2 7.9 85.9 3.9 79.6 0.3 0.8 1.3 99.1

Susan 0 n.m. 2.7 95.8 0.1 95.6 n.d. n.d. <0.1 98.5

silt 7 0.2 2.5 94.6 0.9 93.7 n.d. n.d. n.d. 97.3

loam 62 1.7 2.9 94.7 0.4 94.1 n.d. n.d. 0.1 99.3

120 2.7 2.8 94.3 1.2 92.1 0.1 0.4 0.6 99.8

181 3.4 11.7 81.4 1.3 78.3 0.2 0.2 1.5 96.5

n.m. = not measured n.d. = not detected



Section A7.2.1 and

A7.2.2.1 Annex Point IIIA XII 1.1






1 REFERENCE

Official

use only

1.1 Reference Gilges, M. (2000): Aerobic degradation and metabolism of TI-435 in

four soils.


497/99; amended report of 9 April 2001




letter of access

None


protection




SETAC (1995) and US EPA Subdivision N, 162-1

2.2 GLP Yes

2.3 Deviations No



C]TI-435



C]TI-435

3.1.2 Lot/Batch number [nitroimino-14

C]TI-435: 11553/1

[thiazolyl-2-14

C]TI-435: 11649/4

3.1.3 Specific

radioactivity

[nitroimino-14

C]TI-435: 3.78 MBq/mg

[thiazolyl-2-14

C]TI-435: 3.84 MBq/mg

3.1.4 Purity [nitroimino-14

C]TI-435:



[thiazolyl-2-14

C]TI-435:

Radiochemical purity: > 98% according to radio TLC



microorganisms

No



Section A7.2.1 and







3.2 Reference

substance








NTG: Nitroguanidine


3.3 Test system

3.3.1 Soils The route and rate of degradation was investigated in 3 European soils

and 1 US soil.

3.3.2 Test system

sampling

The soils Laacher Hof and Höfchen were sampled freshly from the field

and the soils BBA 2.2 and Howe were freshly taken from wooden boxes

in which the soils were stored (in open area under grass cover to

maintain their biological activity).

3.3.3 Test system

preparation

A few days before starting the test, the soils were air dried to such an

extend that they could be sieved to a particle size of 2 mm.

Subsequently, the soil moisture was determined. The microbial biomass

was determined immediately before the start and within the course of

the study according to ANDERSON AND DOMSCH (1978).

3.3.4 Test conditions Incubation system: 300 mL Erlenmeyer flasks to which traps for the

adsorption of volatile compounds (CO2 and organic volatiles) were

attached.

Oxygen conditions: aerobic

Light conditions: dark

Temperature: 20 ± 1°C

Soil moisture: 40% of the maximum water holding capacity (Laacher

Hof and Höfchen) or 75% of 333 mbar moisture (BBA 2.2 and Howe)


solution and

application

The test substance (dissolved in acetonitrile) was applied to an aliquot

of about 40 g soil which was then homogeneously mixed into the

remaining total amount of soil. Aliquots corresponding to 100 g of dry

soil were weight into the test vessels.

Soils Laacher Hof, Höfchen and BBA 2.2 were applied with

[nitroimino-14

C]TI-435 and soil Howe with [thiazolyl-2-14

C]TI-435.

3.3.6 Rate of application 13.3 µg a.s./100 g dry soil, equivalent to an annual rate of

approximately 300 g a.s./ha (assuming 15 cm soil depth and 1.5 g/cm3

soil density).

3.3.7 Duration of test 120 days or 365 days



Section A7.2.1 and







3.3.8 Sampling and

extractions

Duplicate or single vessels were processed at 0, 1, 7, 14, 33, 61, 90 and

120 days after treatment. In case of soils BBA 2.2 and soil Howe,

additional sampling intervals were on day 180, 271 and 365 after

treatment.

At each sampling date, the soil was four times extracted with

acetonitrile and the fifth time with water. To obtain some information on

the nature of residues remaining in soil, the soils were additionally

subjected to hot extraction with acetonitrile : water (50:50, v/v) by

refluxing for two hours. The amount of bound residues was calculated

as remaining radioactivity in soil (obtained by combustion after first

extraction) substracted by the radioactivity measure in the second (hot)

extracts. The radioactivity measured in the filters (≤ 2.7% of applied

radioactivity) was added to bound residues.

3.3.9 Biomass

determination

For the purpose of biomass determination at the beginning and at the

end of the study (day 120 for soils Laacher Hof and Höfchen, day 180

and 365 for soil Howe and day 120, 180 and 365 for soil BBA 2.2),

samples with and without active ingredient were incubated under

identical conditions as in the main experiment.


Concentration of test substance and degradation products: TLC on RP-

18 plates and silica gel plates.

LOQ for a single component in the extracts: 0.1% of the applied

radioactivity, corresponding to ca. 0.2 µg/kg soil.


degradation

products

Identified

Degradation products were identified by TLC co-chromatography and

detection in the UV chamber at 254 nm. The identity of all

degradation products was confirmed by HPLC-MS/MS.

4 RESULTS

4.1 Recovery The total recovery of radioactivity on day 0 was taken as 100%. Within

the course of the study, the total recoveries varied between 91.5 and

109. 2% of the applied radioactivity in the silt loam (Laacher Hof),

100.2 and 107.0% in the silt (Höfchen), 97.2 and 101.7% in the

loamy sand (BBA 2.2) and between 96.8 and 101.4% in the sandy

loam (Howe).

4.2 Extracted and

non-extracted

radioactivity

The amount of extracted residues decreased with time in all soils while

bound residues were increasing. The most significant decrease

occurred in soil Höfchen, i.e. from 97.7% on day 0 to 82.5% on

day 120. After 120 days of incubation, the non-extracted radioactivity

reached 5.9 - 9.4% of the applied radioactivity in the German soils

and 5.1% in the US soil.

For details see Table A7_2_1-2.


test substance

After 120 days, between 54.3 and 85.8% of the applied radioactivity

represented TI-435. In the loamy sand and sandy loam (BBA 2.2 and

Howe), which were incubated for 365 days, the amount decreased to

57.8% and 75.8%, respectively.

For details see Table A7_2_1-2.



Section A7.2.1 and







4.4 Degradation rate

4.5 Mineralisation

4.6 Metabolites and

degradation

products

Four metabolites were identified of which only MNG (N-methyl-N’-

nitroguanidine) exceeded 10% of the applied radioactivity in one of the

tested soils (Laacher Hof). TZNG (N-(2-chloro-5-thiazolylmethyl)-N’-

nitroguanidine) amounted to 9.1% in the silt soil (Höfchen) on day 120

with increasing tendency. Maximum amounts of 6.7% and 2.4% were

identified as NTG (Nitroguanidine) and TZMU (N-(2-chloro-5-

thiazolylmethyl)-N’-methylurea), respectively. The amount of

unidentified radioactivity (origin and diffuse radioactivity) did not

exceed 5% of the applied radioactivity.

The degradation rate of MNG (N-methyl-N’-nitroguanidine) could not be

calculated since the highest concentration (10.7% of the applied

radioactivity) occurred at the end of incubation (day 120).

4.7 Degradation route


5.1 Materials and

methods

Aerobic degradation and metabolism of TI-435 was investigated in three

German (silt loam, silt and loamy sand) and one US (sandy loam) soil

according to SETAC (1995) and US EPA Subdivision N, 162-1. No

deviations occurred. The test substance was applied at a

concentration of 13.3 µg a.s./100 g dry soil, equivalent to an annual rate

of approximately 300 g a.s./ha. The soils were incubated in the dark at

20°C for either 120 (silt loam and silt) or 365 (loamy sand and sandy

loam) days.

5.2 Results and

discussion

Total recoveries of applied radioactivity ranged between 91.5 and

109.2%. The amount of extracted radioactivity showed a tendency to

decrease with time due to a slow increase in the amount of bound

residues and due to mineralisation. After 120 days, TI-435 represented

68.6 and 54.3% of the applied radioactivity in Laacher Hof and Höfchen,

respectively, and after 365 days TI-435 represented 57.8 and 75.8% of

the applied radioactivity in BBA 2.2 and Howe, respectively. MNG (N-

methyl-N’-nitroguanidine) was identified as a major metabolite with a

maximum level of 10.7% of the applied radioactivity.

DT50 values for the degradation of TI-435 in soil ranged between 143



Section A7.2.1 and







and 1001 days (first order).


5.3.1 Reliability 1






Date 2007-01-10

Materials and Methods The applicants version is acceptable.

The aerobic degradation and metabolism of TI-435 were investigated in three

German soils and one US soil with an application rate of 300 g a.i./ha. Two

different radiolabels were used. The studies were performed in compliance with

the resp. SETAC Procedures and the US EPA-Guideline §162-1. The soil samples

were incubated in the dark at 20°C with a moisture content of 75% of 1/3 bar (two

soils) resp. 40% MWHC (two soils).

Results and discussion The applicants version is adopted.

In the four aerobic laboratory soil degradation tests, the amount of extracted

radioactivity only slightly decreased with time with up to 86% of unchanged

clothianidin still identified after 120 days.

First order DT 50 values determined for clothianidin in the individual soils range

from 143 days to > 1 year.

A total number of four metabolites were detected in the soil extracts, with MNG

appearing at a max. level of 10.7% after 120 days in one soil. Furthermore, two

metabolites were found at lower levels between 5 and 10% whereas TZMU only

occurred at levels < 5%.

Mineralization was low to negligible accounting for a max. of 11,2% after 120

days resp. max. 14.8% in one soil after 365 days.

The amount of bound residues slightly increased towards the end of the study with

a max. of 9,4% after 120 days resp. 12.8% in one of the four soils after one year.

Conclusion The applicant's summary is acceptable. In light of DT 50 values up to more than

one year due to only minor metabolism and negligible ultimate degradation,

clothianidin shows in an overall assessment high persistence in soil.

Reliability 1


Remarks none

COMMENTS FROM ...







Section A7.2.1 and











Remarks



Soil

Origin

Laacher Hof

Germany

Höfchen

Germany

BBA 2.2

Germany

Howe

USA

Soil type (according to USDA) silt loam silt loamy sand sandy loam


sand 2000-50 µm [%]

silt 50-2 µm [%]

clay < 2 µm [%]

36.9

51.1

12.0

8.5

81.3

10.2

80.5

12.3

7.2

65.7

26.4

7.9


sand 2000-63 µm [%]

silt 63-2 µm [%]

clay < 2 µm [%]

35.9

53.0

11.2

8.2

81.5

10.3

79.7

13.1

7.2

63.5

28.6

7.9

pH (water)

(CaCl2)

8.1

7.3

7.8

7.2

6.0

6.3

6.7

6.7

organic carbon [%]

organic matter [%]

0.9

1.5

2.7

4.6

2.5

4.3

1.1

1.9

CEC [meq/100 g soil] 8 15 10 10

Microbial biomass*




216

222

n.d.

552

476

n.d.

285

259

182

166

n.d.

81

40% WHCmax [g water to 100g dry soil]

75% of 333 mbar moisture

[g water to 100 g dry soil]

14.6

20.7

25.3

n.d.

18.0

16.1

13.7

14.8

Pesticide use history no prior use of

TI-435**

no prior use of

TI-435 or other

chloronicotinyl

compounds

no prior use of

TI-435 or other

chloronicotinyl

compounds

no prior use of

TI-435 or other

chloronicotinyl

compounds

* On day 0 determined in soil without active substance. On day 120 and 365 determined in soil containing the active substance.

** Imidacloprid was used as a seed dressing on sugar beets in April 1996 (<120 g/ha).




Table A7_2_1-2: Recovery of radioactivity and distribution of the active substance and metabolites after

application of [14C]TI-435 to 4 different soils and aerobic incubation at 20°C (values given

in % of applied radioactivity)

Soil/

label

position

Days

after

appli-

cation

14CO2 Non-

extracted

radio-

activity

Extracted

radio-

activity

Origin

TI-435 TZNG

TZMU

MNG

NTG

Diffuse

radio-

activity

Total

Laacher 0 n.m. 2.1 98.0 0.4 92.6 n.d. n.d. n.d. n.d. 5.0* 100

Hof 1 <0.1 2.0 101.9 0.3 100.7 n.d. n.d. n.d. n.d. 0.9 103.9

silt loam/ 7 0.2 2.8 106.2 2.7 98.5 0.7 0.9 1.0 n.d. 2.3 109.2

14 0.4 2.9 99.6 2.3 90.2 1.0 1.1 2.1 0.1 2.9 102.8

nitroimino 33 1.1 5.0 95.1 0.3 84.0 2.6 2.2 5.2 0.2 0.6 101.3

61 2.2 3.8 85.5 0.4 71.3 3.3 2.3 6.3 1.2 0.6 91.5

90 3.8 4.4 95.4 0.4 74.6 4.5 2.2 8.9 3.2 1.6 103.6

120 5.1 8.5 92.5 0.3 68.6 5.1 2.4 10.7 3.7 1.8 106.1

Höfchen 0 n.m. 2.4 97.7 0.3 92.0 n.d. n.d. n.d. n.d. 5.5* 100

silt/ 1 0.1 2.1 103.9 0.4 102.7 n.d. n.d. n.d. n.d. 0.8 106.1

7 0.4 3.9 102.6 0.6 97.0 2.0 0.9 1.7 n.d. 0.5 107.0

nitroimino 14 1.0 3.3 98.6 2.2 88.4 3.0 1.2 3.1 0.2 0.5 102.8

33 3.1 5.1 95.5 0.4 79.4 5.8 1.9 6.1 0.7 1.1 103.8

61 6.0 6.0 88.2 0.5 68.7 7.2 1.5 6.6 2.7 1.0 100.2

90 8.8 7.5 88.1 0.5 64.2 8.2 1.0 8.2 5.3 0.7 104.4

120 11.2 9.4 82.5 0.3 54.3 9.1 1.1 9.5 6.7 1.7 103.1

BBA 2.2 0 n.m. 2.0 98.1 0.4 93.6 n.d. n.d. n.d. n.d. 4.1* 100

loamy 1 <0.1 2.2 95.4 0.5 94.2 n.d. n.d. n.d. n.d. 0.6 97.7

sand/ 7 0.2 2.9 95.9 0.5 92.8 0.4 0.3 1.3 n.d. 0.6 99.0

14 <0.1 2.7 98.9 2.8 92.3 0.5 0.8 1.6 0.3 0.6 101.7

nitroimino 33 1.2 4.4 91.7 0.1 84.3 2.1 1.1 3.3 0.1 0.5 97.2

61 2.4 5.2 91.4 0.6 82.3 2.4 0.9 3.6 1.0 0.7 98.9

90 3.5 5.0 91.7 0.4 77.2 3.3 1.2 5.2 2.7 1.6 100.2

120 4.7 5.9 90.0 0.3 73.3 4.4 1.2 5.9 3.4 1.5 100.5

180 6.7 7.9 85.5 0.5 69.1 4.8 0.8 5.1 3.9 1.5 100.0

271 9.2 11.2 78.5 0.4 60.3 5.2 0.8 5.2 5.2 1.4 98.8

365 11.3 12.8 77.1 0.3 57.8 6.0 0.7 5.4 6.5 0.5 101.2

Howe 0 n.m. 1.8 98.2 0.4 93.6 n.d. n.d. - - 4.3* 100

sandy 1 0.1 1.7 95.2 0.4 93.5 <0.1 n.d. - - 1.3 97.0

loam/ 7 0.7 2.6 97.6 0.4 96.3 0.4 0.2 - - 0.3 100.7

14 1.3 2.5 97.7 1.2 95.6 0.2 0.2 - - 0.6 101.4

thiazolyl 33 2.5 3.2 93.8 0.4 91.9 0.9 0.1 - - 0.6 99.5

61 3.8 2.7 91.0 0.5 88.2 1.3 0.3 - - 0.7 97.5

90 5.5 4.1 90.3 0.9 86.7 1.5 0.3 - - 1.0 99.9

120 6.9 5.1 89.1 0.2 85.8 1.7 0.3 - - 1.3 101.2

180 9.0 5.0 86.0 0.4 81.9 1.9 0.3 - - 1.5 99.9

271 12.1 5.5 79.3 0.7 75.4 2.3 0.2 - - 0.7 96.8

365 14.8 6.6 79.0 0.5 75.8 2.5 0.2 - - 0.2 100.4

n.m. = not measured n.d. = not detected

* The radioactivity in the hot extracts was added to Diffuse Radioactivity since no identification by TLC was made.




Field soil dissipation and accumulation

1 REFERENCE

Official

use only

1.1 Reference Schramel, O. (2000b): Dissipation of TI-435 (600 FS) in soil under field

conditions (France, Germany, Great Britain).

Bayer AG, 51368 Leverkusen, Germany; unpublished report no. RA-

2065/98


1.2.1 Data owner Sumitomo Chemical Takeda Agro Co., Ltd. / Bayer CropScience


letter of access

No


protection

Data on existing active substance for first entry to Annex I.



SETAC (1995) and ECPA Guidance Document on Field Soil Dissipation

Studies, D/97/NM/2047 (1997)

2.2 GLP Yes

2.3 Deviations Deviations (to SETAC): No crops were grown.


3.1 Test material TI-435 (600 FS)


3.1.2 Type of

formulation

FS (flowable concentrate for seed treatment)

3.1.3 Content of a.s. (TI-

435)

620.0 g/L


properties

a.s. not inhibitory to microorganisms

a.s. not volatile (vapour pressure: 1.3 · 10-10

Pa)

3.1.5 Stability of the test

substance

The guidelines of formulation development ensure that on a chemical

and physical basis the test substances and their dilutions (as spray liquid)

correspond to the international requirements and have sufficient stability.

3.1.6 Method of analysis Analysis according to method 00540 (as described in section A4.2)

based on liquid chromatography with electrospray MS/MS detection.

The limit of quantification (LOQ) and the limit of detection (LOD) for

TI-435 are 5 µg/kg soil and 2 µg/kg soil, respectively.

3.2 Degradation

products

Apart from the parent compound, soil samples were analysed for the

degradation products:




for degradation

products

As described in 3.1.6. The limit of quantification (LOQ) for MNG and

TZNG is 5 µg/kg soil both. The limit of detection (LOD) for MNG and

TZNG is 2 µg/kg soil both.

3.3 Reference

substance

The parent compound and the degradation products TZNG and MNG

were used as reference substances for co-chromatography.

Isotopically labelled internal standards (d3-TI-435, 13

C, 15

N-TZNG and






for reference

substance

3.4 Test system To determine the extent of dissipation of TI-435

in Northern European soils, four trials were settled in typical agricultural

regions with different climates and soil types in Germany

, the UK and France . They

were conducted without vegetation.

3.5 Soil types


3.6.1 Test substance

application

A single spray application was made on bare soil in spring 1998 at a rate

of equivalent 150 g a.s./ha with 300 L/ha water (plot sizes: 225 to

360 m2).

3.7 Sampling and

work-up

3.7.1 Soil sampling Samples were taken immediately after drying of the spray and at

11 intervals thereafter. The last samples were taken after about two years

(725 to 750 days after application). From all trials, 20 treated and at least

10 control samples were taken (cores of 5 cm in diameter). The sampling

spots were distributed statistically over the plots to get representative

samples. Samples were deep frozen (≤ -18°C) until analysis.

3.7.2 Sample work-up Soil samples were extracted with a mixture of acetonitrile/water/acetic

acid (200:800:0.8, v/v/v). Thereafter, internal standard was added and

the extract analysed as described in 3.1.6.

4 RESULTS

4.1 Controls All concentrations in the control samples were below the limit of

detection except for one sample with MNG residues being between the

LOD and the LOQ (UK trial, day 0).

4.2 Total residues The total residues in the 0-10 cm soil layer ranged from 98.7-130 µg/kg

soil on day 0 and decreased to 7.3-38.4 µg/kg until the end of the study

after two years. In the 10-20 cm soil layer, the total residues remained

between the LOQ and LOD and in the 20-30 cm layer below 2 µg/kg

(LOD) during the test period.

4.3 Residues of TI-435 On day 0, the concentration of TI-435 ranged from 98.7 to 128 µg/kg

soil in the 0-10 cm layer. At the end of the study after 2 years, 7.3 to

35.8 µg TI-435/kg soil were detected. No translocation of the active

substance into deeper soil layer could be observed in any trial. In the 10-

20 cm soil layer, concentrations remained below the LOQ and in the

20-30 cm layer below the LOD.

4.4 Residues of MNG The metabolite MNG could be detected in the 0-10 cm soil layer in

concentrations between the LOQ and LOD at 3 trial locations (total of 15

samples). No residues could be detected in deeper soil layers.

4.5 Residues of TZNG The metabolite TZNG was detected above the LOQ in one single sample

(UK, day 479) at a concentration of 6.1 µg/kg in the 0-10 cm soil layer.

In the other three trials, TZNG was not detected in any soil layer during

the whole test period.

4.6 Degradation of DT50- and DT90-values are presented in Table A7_2_2_2-2.





total residues in

soil

4.7 Degradation of

TI-435 in soil

DT50- and DT90-values are presented in Table A7_2_2_2-3.


5.1 Materials and

methods

The field soil dissipation and accumulation of TI-435 was studied

according to SETAC (1995) and ECPA Guidance Document on Field

Soil Dissipation Studies, D/97/NM/2047 (1997). Deviating from

SETAC, no crops were grown.

5.2 Results and

discussion

After 24 months of test duration, a mean of 19% of the applied amount

based on total residues was recovered from the soil. Total residues were

recovered with half-life periods of 19 to 362 days, with a mean value of

135 days.

After 24 months of tests duration, a mean of 19% of the applied amount

based on the active substance was recovered from the soil. The active

substance TI-435 was degraded with half-life periods of 16 to 258 days,

with a mean value of 103 days.

Translocation of TI-435 into deeper soil layers than 10-20 cm can be

excluded down to a concentration of 2 µg/kg corresponding to less than

2% of the initial concentration of the active substance.

The only exception was the UK trial , where residues

could be found in the 0-10 cm layer. No

translocation into soil layers below 20 cm was observed.

5.3 Conclusion TI-435 is persistent in soil.

5.3.1 Reliability 1









EVALUATION BY RAPPORTEUR MEMBER STATE (*)

Date 2007-01-10

Materials and Methods The applicant´s version is acceptable. In this study conducted in compliance with the

requirements of the guidelines stated, the dissipation behaviour and the translocation

of TI-435 into deeper soil layers was determined in soil without vegetation under field

conditions. Four trials were located in typical agricultural regions in Germany, Great

Britain and France. A single spray application of TI-435 (600FS) to bare soil was

conducted between March and May 1998. The application rate was 150 g a.i./ha for all

trials.

Results and discussion The applicant´s version is acceptable with the exception of the DT50 calculation. In all

eight trials the concentrations of the total residues of clothianidin in soil declined with

time. All trials have been evaluated with both SFO and FOMC-kinetics. The

respective DT50 and DT90-values are given in table 1. Since the fit for SFO-kinetics is

very poor (r2 ranging from 0.69 to 0.88) an additional FOMC evaluation has been

performed. Although the optical check is acceptable, no chi2 values have been

documented due to FOCUS-kinetics report.

Therefore a pragmatic approach has been chosen in the risk assessment (DOC IIA) to

derive DT50-values for PEC-calculations.

. Table 1: Degradation of clothianidin under field conditions. DT50 values based on 2

models and normalised to 12 and 20ºC, respectively

clothianidin

field data

Mean soil

temp *, ºC

fitted SFO model fitted FOMC model

DT50

(d) DT50(12º) DT50(20º) DT50 (d)

DT50

(12º)

DT50

(20º)

D, bare soils 8.9 32 25 13 16 12 7

D 10.6 165 148 79 76 68 36

UK 10.5 506 449 239 258 229 121

F 11.7 240 234 125 62 61 32

UK. crop.

soils 10.5 394 349 186 189 168 88

F 10.6 263 235 125 29 26 14

F 17.6 369 578 305 126 197 104

ES 18.5 219 368 195 82 138 73

Arithmetric

mean 12.4 273 298 157 105 112 59

Geometric

mean 12.0 219 226 120 76 78 41

*: Temperature is not cited in Docs, extracted from Pflanzenschutz-Nachrichten Bayer

56/2003, 1, p.68.

Conclusion The half-lives confirm that TI-435 is persistent in soil also under field conditions. The

validity criteria are fulfilled.

Reliability 1






Remarks In a separate report (MR-414/00, dated 2000/9/20) the author recalculated the DT50

values (fitted FOMC Model) for clothianidin on the experimental basis of the study

evaluated above to fit a simple first order model (fitted SFO Model) using the

modelling software package ModelManager. The resulting DT50 values varied

between 32 – 506 days. In a second step, based on the average soil temperatures these

recalculated SFO DT50 values were transformed to the reference temperature of 20°C

(Q10 = 2.2). This conversion results in DT 50 values for clothianidin in the four bare

soil trials tested in a range from 13 – 239 days.

Hence, depending on the calculation method large variations can be observed between

the calculated half-lives!

The applicant has announced that considering the DT 90 (f) > 1 year and the DT 50 (f)

> 3 months, a soil accumulation study in two soil types with test sites in F, UK and D

is ongoing, with a final report to be expected in 2005.

COMMENTS FROM ...

Date



Conclusion

Reliability

Acceptability

Remarks



Table A7_2_2_2-1: Soil characteristics (0-30 cm layer)

Soil

Origin

Burscheid

Germany

Monheim

Germany

Bury St.

Edmunds, UK

Guiseniers

France

Soil type (according to USDA) silty loam sandy loam sandy loam silt loam


sand 2000-50 µm [%]

silt 50- 2 µm [%]

clay < 2 µm [%]

15.9

67.3

16.8

68.1

21.5

10.4

63.6

19.9

16.5

12.9

76.0

11.1


sand 2000-63 µm [%]

silt 63- 2 µm [%]

clay < 2 µm [%]

13.2

70.0

16.8

65.2

24.4

10.4

61.6

21.9

16.5

8.1

80.8

11.1

pH (CaCl2)

(KCl)

6.25

6.45

6.32

6.80

7.45

7.96

5.90

5.94

organic carbon [%]

organic matter [%]

0.97

1.67

0.89

1.53

0.86

1.48

1.16

2.0

CEC [meq Ba/100 g dry soil] 15 10 13 13

moisture capacity [g/100 g dry soil] 37.5 29.6 34.3 48.1

Table A7_2_2_2-2: Degradation of total residues of TI-435 in soil

Location Soil layer [cm] DT50 [days] DT90 [days] Order of function

Germany

(Burscheid)

0-10 19 345 1st order multi

compartment model

Germany

(Monheim)


compartment model

Great Britain (Bury

St. Edmunds)

0-10 362 * 1st order multi

compartment model

France

(Guiseniers)


compartment model

Mean value 135 -

* Calculated DT90-values of >>1000 days were neglected (Model Manager, Version 1.1, Cherwell Scientific

Ltd., Oxford, UK).

Table A7_2_2_2-3: Degradation of TI-435 in soil

Location Soil layer [cm] DT50 [days] DT90 [days] Order of function

Germany

(Burscheid)


compartment model

Germany

(Monheim)


compartment model

Great Britain (Bury

St. Edmunds)


compartment model

France

(Guiseniers)


compartment model

Mean value 103 -

* Calculated DT90-values of >>1000 days were neglected (Model Manager, Version 1.1, Cherwell Scientific

Ltd., Oxford, UK).


Section A7.2.2.3 Annex Point AIII XII.1.4

Extend and nature of bound residues





-




Date


Conclusion

Remarks






Remarks




tion A7.2.2.4

Annex Point AIII XII.1.4

Other soil degradation studies

The rate and route of degradation of a metabolite

1 REFERENCE

Official

use only

1.1 Reference Dorn, R. (2000): Degradation of 14

C-MNG, a degradate of TI-435, in

three different soils.

SLFA 67435 Neustadt/Weinstrasse, Germany; unpublished report no.

TAK06




letter of access

None


protection




SETAC (1995)

2.2 GLP Yes

2.3 Deviations None (to SETAC)



C]MNG


C]MNG

3.1.2 Lot/Batch number radiolabelled MNG: 14627623

3.1.3 Specific

radioactivity

1.730 MBq/mg

3.1.4 Radiochemical

purity

96.2% according to radio TLC

97.1% according to radio HPLC


microorganisms

No

3.2 Reference

substance

None

3.3 Test system

3.3.1 Soils The rate of degradation was investigated in 3 German soils (Laacher

Hof AXXa, Laacher Hof AIII and Höfchen am Hohenseh).

For soil characteristics see Table A7_2_2_4/01-1.

3.3.2 Test system

sampling

The soils were sampled freshly from the field less than 3 months before

start of the test.

3.3.3 Test system

preparation

After arrival at the test facility, the soils were carefully air-dried and

sieved to a particle size of 2 mm. 100 g dry soil were weighed into

each test vessel and soil moisture was adjusted to approximately 48% of

MWHC. Then the vessels were closed with a cotton wool stopper and

pre-incubated at 20 2°C in the dark for five days. The microbial

biomass was determined immediately before the start and at the end of

the test period according to ANDERSON AND DOMSCH (1978).

3.3.4 Test conditions

attachment (containing soda lime and oil wetted quartz wool) that



tion A7.2.2.4




permitted some gas exchange but which adsorbed potential 14

C-volatiles

including CO2;

Oxygen conditions: aerobic;


Temperature: 20 ± 1°C;

Soil moisture: 48% of the maximum water holding capacity.

3.3.5 Rate of application 9.5 µg/100 g dry soil


solution and

application

A stock solution was prepared by dissolving the test substance in

acetonitrile:water (1:1, v/v) to a final concentration of 1.787 mg/mL.

The test substance was to be applied to the soil in distilled water. An

appropriate volume of the stock solution was transferred into a

volumetric vessel, the organic solvent was evaporated under a gentle

stream of N2 and the vessel was made up to volume with distilled water.

Appropriate amounts of the aqueous application solution were pipetted

in small drops to the surface of the pre-incubated soil samples. The test

systems were closed with the trapping system for volatiles and

incubated under appropriate conditions.

3.3.7 Control of moisture

content

The test vessels were weighed monthly. A loss of soil moisture was

balanced by the addition of appropriate amounts of distilled water.

3.3.8 Duration of test 126 days x

3.3.9 Sampling and

extractions

Duplicate or single vessels were processed on 0, 1, 7, 14, 33, 61, 90 and

120 days after treatment.

At each sampling date, the soil was extracted three times with

acetonitrile, followed by one extraction with 0.01M CaCl2. Then

residual radioactivity in soil was determined via LSC following

combustion of soil aliquots. To obtain information on the nature of

residues remaining in soil, the soils were additionally subjected a

Soxhlet extraction with acetonitrile/water (1:1, v/v).

3.3.10 Biomass

determination

For the purpose of biomass determination at the beginning and at the

end of the study period, samples without (day 0) and with active

substance (day 120) were prepared and incubated under identical

conditions as in the main experiment.


Concentration and characterization of the application solution:

HPLC; pre- and main column filled with Superspher 60 RP Select B;

gradient of 2 solvents: (A) acetic acid (1%, v/v) + 1 g/L ammonium

acetate in water, and (B) acetonitrile; flow rate: 1.0 mL/min.

Identification of test substance and degradation products in extracts:

a) organic extract: TLC on Silica gel RP18 F254 plates; mobile phase:

methanol:acetone:water (10/10/80, v/v/v);

b) CaCl2 extract: TLC on Silica gel 60 F254 plates; mobile phase: 1-

butanol/H2O/acetone/glacial acetic acid (65/10/5/15, v/v/v/v);

c) soxhlet extract: TLC on Silica gel 60 WF254; mobile phase: 1-

butanol/H2O/acetone/glacial acetic acid (65/10/5/15, v/v/v/v).

LOD: 125 dpm per lane equivalent to 7222 counts/mm2.

3.3.12 Degradation

products

Not identified.



tion A7.2.2.4




4 RESULTS

4.1 Recovery Nine aliquots of the application solution were measured by LSC during

the application procedure and the mean (9.52 g/test system) was taken

as the applied amount (100%) for all further data evaluations.

Within the course of the study, the total recoveries varied between 95.05

and 97.22% of the applied radioactivity in the sandy loam (Laacher Hof

AXXa), 90.18 and 97.66% in the silt loam (Laacher Hof AIII) and

between 94.19 and 97.67% in the silt (Höfchen).

4.2 Extracted and

non-extracted

radioactivity

The amount of extractable residues decreased with time in all soils due

to the increase in bound residues and mineralisation. The most

significant decrease occurred in soil Höfchen, i.e. from 96.74% on day 0

to 63.13% on day 120, followed by soil Laacher Hof AIII (97.12 to

66.24%) and soil Laacher Hof AXXa (96.74 to 80.14%). After 120 days

of incubation, the non-extractable radioactivity amounted to 14.94,

16.27 and 11.01% of applied radioactivity in the three soils,

respectively.

For details see Table A7_2_2_4/01-2.


test substance

After 120 days, 40.60, 43.53 and 34.60% of the applied radioactivity

represented MNG in soil Laacher Hof AXXa, soil Laacher Hof AIII and

soil Höfchen, respectively.


4.4 Mineralisation After 120 days of incubation, 5.39, 11.75 and 16.60% of the applied

radioactivity were mineralised to 14

CO2 in Laacher Hof AXXa, Laacher

Hof AIII and Höfchen soil, respectively. At one sampling date, a

negligible amount of other volatile products was found in Höfchen soil.

4.5 Metabolites and

degradation

products

Identification of degradation products was not done. However, the

portion of non-identified radioactivity increased continuously in soil

Laacher Hof AXXa to a maximum of 39.55% of applied radioactivity on

day 120. In soil Laacher Hof AIII and Höfchen not identified

radioactivity amounted to 18.41% and 24.76% of applied radioactivity at

the end of the test period, whilst the maximum amount of 22.35 and

28.56% of applied radioactivity in these soils was reached on day 33 and

day 61, respectively.


4.6 Degradation rate For the degradation of MNG in soil the following DT50 values were

calculated based on simple first order kinetics:

86.4 days

108.0 days

82.4 days

4.7 Route of

degradation

Not applicable since identification of degradation products was not done.


5.1 Materials and

methods

Aerobic degradation of MNG was investigated in three German soils

(sandy loam, silt loam and silt) according to SETAC (1995). No

deviations to SETAC occurred. The test substance was applied on top of

the soil at a concentration of 9.52 µg/100 g dry soil. The soils were

incubated in the dark at 20°C for 120 days.



tion A7.2.2.4




5.2 Results and

discussion

DT50 values for the degradation of MNG in soil were calculated

according to simple first-order model and were 86.4, 108.0 and 82.4 days

for the sandy loam, the silt loam and the silt soil, respectively.


5.3.1 Reliability 1






Date 2007-01-10

Materials and Methods applicants version is acceptable

Comment:

3.3.8 Duration of test: 120 days

Results and discussion applicant's version can be adopted.

In the aerobic laboratory soil degradation test with MNG in 3 soils, total

extractable radioactivity only slightly decreased with time down to levels

between 63.1 – 80.1% after 120 days. At study end, unchanged MNG still

represented 34.0 - 43.5% of the applied radioactivity, the formation of in this

study not further identified radioactivity varied between 18.4 – 39.6%. The

amount of non-extractable residues increased during the course of the study to

levels between 11 – 16.3%after 120 days. Mineralization observed after 120

days accounted for 5.4 – 16.6%. First order DT 50 values determined for MNG

in the individual soils range from 82.4 – 108 days.

Conclusion The applicant's summary and conclusion is acceptable.

The metabolite MNG indicates DT50 values up to 108 days and a low level of

ultimate biodegradation together with the formation of a moderate plateau of

bound residues.

Reliability 1


Remarks none

COMMENTS FROM ...







tion A7.2.2.4








Remarks



Table A7_2_2_4/01-1: Characteristic of the soils

Soil

Origin

Laacher Hof

AXXa

Laacher Hof

AIII

Höfchen am

Hohenseh

Soil type (according to USDA) sandy loam silt loam silt


sand 2000-50 µm [%]

silt 50-2 µm [%]

clay < 2 µm [%]

72.4

22.6

5.0

36.9

51.1

12.0

8.5

81.3

10.2


sand 2000-63 µm [%]

silt 63-2 µm [%]

clay < 2 µm [%]

n.d.

n.d.

n.d.

35.9

53.0

11.2

8.2

81.5

10.3

pH (water)

(CaCl2)

7.17

6.32

7.88

6.72

7.30

6.52

organic carbon [%]

organic matter [%] = OC * 1.72

1.02

1.75

0.98

1.69

1.55

2.67

CEC [meq/100 g soil] 8 8 15

Microbial biomass*



365

239

276

190

389

292

WHCmax [g water to 100g dry soil] 34.42 36.40 63.10

* On day 0 determined in soil without active substance, on day 126 determined in soil containing the active

substance.




Section A7.2.3.1/01 Annex Point IIIA XII.1.2

Adsorption and desorption of metabolites and degradation products

1 REFERENCE

Officialuse only

1.1 Reference (2000):


1.2.1 Data owner




2.1 Guideline study Yes OECD 106 (1981), US EPA Subdivision N, § 163-1 (1982) and Environmental chemistry and fate guidelines for registration of pesticides in Canada: Trade Memorandum T-1-255, Section 6.2B

2.2 GLP Yes

2.3 Deviations No


3.1 Radiolabelled test material

3.1.1 Molecular formula

3.1.2 Molecular weight


3.1.4 Specific activity

3.1.5 Purity


None

3.1.7 Method of analysis HPLC with Radio-HPLC-Detector and UV-Detector

3.2 Degradation products


3.3.1 Method of analysis for reference substance





3.4 Soil types


3.5.1 Test system

3.5.2 Test solution and Test conditions


3.6.1 Preliminary test

3.6.2 Screening test: Adsorption

3.6.3 Screening test: Desorption

3.6.4 HPLC-method According to ”OECD-HPLC-method”: No


4 RESULTS


4.2 Screening test: Adsorption

4.3 Screening test: Desorption

4.4 Material balance





4.5 Calculations

4.5.1 Ka , Kd

4.5.2 Kaoc , Kdoc

4.6 Degradation product(s)



Adsorption and desorption of [14C]MNG were measured using a batch equilibrium procedure according to OECD 106. The guideline is fulfilled, no relevant deviations from the guideline occurred.


The Kaoc–values varied between 5.2 and 34.3 and the Kdoc–values between 13.0 and 44.0.

5.2.1 Adsorbed amount [%]

5.2.2 Ka 0.02 -0.37 mg/g

5.2.3 Kd 0.15 -0.48 mg/g

5.2.4 Kaoc 5.2 -34.3 mg/g (mean: 20.5 mg/g) x

5.2.5 Ka/Kd 0.77 , 1.27 , 0.72

5.3 Conclusion Based on the classification of MCCALL ET AL. (1980), MNG is classified as being very highly mobile in soil.

5.3.1 Reliability 1








Date 2005-02-02



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks




Adsorption and desorption of metabolites and

degradation products

1 REFERENCE

Official

use only

1.1 Reference Möndel, M. and Hein, W. (2000): Adsorption/desorption of 14

C-TZNG, a

degradate of TI-435, on five different soils.

SLFA, 67435 Neustadt/Weinstrasse, unpublished report of study TAK01




letter of access

None


protection




OECD 106 (1981), US EPA Subdivision N, § 163-1 (1982) and

Environmental chemistry and fate guidelines for registration of

pesticides in Canada: Trade Memorandum T-1-255, Section 6.2B

2.2 GLP Yes

2.3 Deviations No


3.1 Radiolabelled test

material

[14

C]TZNG

(= [14

C] N-(2-chloro-5-thiazolylmethyl)-N’-nitroguanidine)

TZNG is a degradate of TI-435 in soil.

3.1.1 Molecular formula C5H6ClN5O2S

3.1.2 Molecular weight 235.7 g/mol



3.1.5 Purity Radiochemical purity: 97.9% (TLC Radiochromatography)

97.4% (HPLC Radiochromatography)

Chemical purity: 98.7% (HPLC-UV Chromatography)


properties

None

3.1.7 Method of analysis HPLC with Radio-HPLC-Detector and UV-Detector

3.2 Degradation

products

Degradation products tested: No

3.3 Reference

substance Non-labelled TZNG was used as reference substance for co-

chromatography.

Lot no.: 89066524

Chemical purity: 98.6%


for reference

substance

See 3.1.5

3.4 Soil types







3.5.1 Test system Adsorption and desorption of TZNG was measured using a batch

equilibrium procedure to determine Koc values of [14

C]TZNG in three

US and two European soils.

The soils were air-dried, 2 mm sieved and stored at room temperature

until use. Before defined amounts of soil were weighted into the test

vessels (Borosilicate glass centrifuge tubes of 42 mL), the residual

moisture contents of the stored soils were determined and taken into

account.

[14

C]TZNG was dissolved in a 0.01 M CaCl2 solution and applied to the

soils.

3.5.2 Test solution and

Test conditions

The test substance TZNG was tested in a concentration range of 0.04 to

5.0 mg/L. The tests were performed in the dark at 20±1°C.


3.6.1 Preliminary test According to ”OECD 106”: Yes


Adsorption

According to ”OECD 106”: Yes

A volume of 20 mL CaCl2 solution containing nominal concentrations of

0.04, 0.2, 1.0 and 5.0 mg TZNG/L was added to the soils (equivalent

to 7g dry weight for the Elder soil and 12g for the other soils). Soil

and supernatant were separated by centrifugation after the respective

shaking periods on a rotary shaker. Each soil and concentration was

tested in duplicate.


Desorption

According to ”OECD 106”: Performed

One desorption step was carried out at concentrations of 0.04, 0.2 and

1.0 mg/L, while 3 serial desorption steps were performed at 5.0 mg/L.

For this, the application solution was replaced by a fresh 0.01 M CaCl2

solution and shaken vigorously for 24 hours. Soil and supernatant

were separated by centrifugation on a rotary shaker. Each soil and

concentration was tested in duplicate.

3.6.4 HPLC-method According to ”OECD-HPLC-method”: No


4 RESULTS

4.1 Preliminary test In preliminary tests, a soil/solution ratio of 1:1.67 (w/v) and an

equilibration time of 24 hours were determined to be used in the

definitive test. However, for the Elder soil, a soil/solution ratio of

1:2.86 (w/v) was determined to be used in the definite test.

[14

C]TZNG was analysed in the supernatant after 24, 48, and 72 hours

shaking period and found to be stable (95% unchanged test

compound).

4.2 Screening test:

Adsorption

The amount adsorbed to the soils ranged from 22.7 to 34.8% for soil

Quincy, 57.8 to 82.3% for soil Elder, 59.6 to 81.2% for soil Crosby,

55.3 to 78.8% for soil Laacher Hof and 26.6 to 48.7% for soil

BBA 2.1.

4.3 Screening test:

Desorption

The proportion of adsorbed [14

C]TZNG desorbed from the soils ranged

from 14.4 to 30.9% for soil Laacher Hof, from 31.9 to 49.3% for soil

BBA 2.1, from 14.5 to 29.7% for soil Crosby, from 13.7 to 30.4% for

soil Elder and from 45.2 to 55.2% for soil Quincy.

4.4 Material balance The recovery rate of applied radioactivity varied between 92.1% and






162.0% for the five soils and four concentrations.

4.5 Calculations

4.5.1 Ka , Kd Calculations of adsorption and desorption constants are given in Table

A7_2_3_1_02-2

4.5.2 Kaoc , Kdoc Calculations of adsorption and desorption constants are given in Table

A7_2_3_1_02-2

4.6 Degradation

product(s)

The test substance was found to be stable for the duration of the test.


5.1 Materials and

methods

Adsorption and desorption of [14

C]TZNG were measured using a batch

equilibrium procedure according to OECD 106. The guideline is

fulfilled, no relevant deviations from the guideline occurred.

5.2 Results and

discussion

The Kaoc–values varied between 204.5 and 432.5 and the Kdoc–values

between 270.8 and 527.2.

There was good correlation between the concentrations adsorbed and in

solution for the concentration range tested (r > 0.99) in all soils.

5.2.1 Adsorbed amount

[%]

The percentage adsorption of test substance varied between 22.7 and

82.3% of the applied amount depending on soil type and concentration.

5.2.2 Ka 0.63 - 4.71 mg/g

5.2.3 Kd 0.83 - 5.75 mg/g

5.2.4 Kaoc 204.5 - 432.5 mg/g (mean: 275.4 mg/g) x

5.2.5 Ka/Kd 0.76 - 0.88 (mean: 0.81)

5.3 Conclusion Based on the classification of MCCALL ET AL. (1980), TZNG is classified

as being moderately mobile in soil.

5.3.1 Reliability 1









Date 2005/02/02

Materials and Methods The applicant’s version is acceptable d.

Results and discussion Despite minor deficiencies the applicant’s version is acceptable.

Comments:

Items 4.5 and 5.2:

The applicant does not provide the unit of the adsorption/desorption coefficients

Ka and Kd as well as of the organic-carbon normalized adsorption/desorption

coefficients Kaoc and Kdoc. The correct unit is cm3 g

-1.

Items 5.2.2, 5.2.3, and 5.2.4:

The applicant does provide an incorrect unit of mg g-1

for Ka and Kd as well as for

Kaoc. The correct unit is cm3 g

-1.

Conclusion The applicant’s version is acceptable.

Reliability 1

Acceptability Despite minor deficiencies the original study and the study summary are

acceptable.

Remarks

COMMENTS FROM ...









Remarks



Table A7_2_3_1_02-1: Classification and physico-chemical properties of soils used as adsorbents

Quincy Elder Crosby Laacher Hof BBA 2.1

Origin Ephrata,

WA, USA

Watsonville,

CA, USA

New Holland,

OH, USA

Monheim,

Germany

Jockgrim,

Germany

Sand (2000 - 50 µm) 92 75 19 72 90

Silt (50 – 2 µm) 6 16 57 23 8

Clay (< 2 µm) 2 9 24 5 2

Classification (acc. to USDA) sand sandy loam silt loam sandy loam sand

organic carbon [%] 0.2 1.1 1.2 1.0 0.4

organic matter [%] 0.4 1.9 2.0 1.8 0.7

pH (water) 6.7 6.9 7.0 7.2 5.9

pH (CaCl2) 6.2 6.5 7.0 6.3 5.6

CEC [meq/100 g] 8 13 20 8 5

CEC = cation exchange capacity

Table A7_2_3_1_02-2: Adsorption and desorption constants of [14

C]TZNG in five different soils

Soil Adsorption Desorption Mobility*

Ka Kaoc 1/n Kd Kdoc 1/n Kddes (serial)**

Quincy 0.63 261.4 0.9010 0.83 346.2 0.8975 0.75 moderate

Elder 4.71 432.5 0.7832 5.75 527.2 0.7924 5.49 moderate

Crosby 2.84 242.6 0.8070 3.23 276.2 0.8059 3.21 moderate

Laacherhof 2.41 236.0 0.8003 2.86 280.6 0.7924 2.94 moderate

BBA 2.1 0.78 204.5 0.8059 1.03 270.8 0.7923 1.09 moderate

MEAN - 275.4 - - 340.2 - -

* classification according to MCCALL ET AL. (1980)

** serial desorption at 5 mg/L according to the Canadian guideline




Mobility in at least three soil types and where relevant

mobility of metabolites and degradation products

1 REFERENCE

Official

use only

1.1 Reference Stupp, H.P. (2001b): Degradation and translocation behavior of the

insecticide active ingredient TI-435 under field conditions in a lysimeter

(autumn application).

Bayer AG, 51368 Leverkusen, Germany; unpublished report no.

MR051/01


1.2.1 Data owner Sumitomo Chemical Takeda Agro Co., Ltd. and Bayer CropScience


letter of access

No


protection




Germany BBA Part IV, 4-3

2.2 GLP Yes

2.3 Deviations No major deviations



C]TI-435

3.1.1 Radiolabelling Thiazolyl-[methylene-14

C]TI-435


3.1.3 Specific radioactivity 4.51 MBq/mg

3.1.4 Purity Radiochemical purity: > 98% according to radio TLC

Chemical purity: > 99% according to HPLC


properties

TS not inhibitory to microorganisms

TS not volatile (vapour pressure: 1.3 · 10-10

Pa)

Water solubility (20°C): 327 mg/L (see Morrissey & Kramer, 2000a)

3.1.6 Method of analysis Analysis by TLC methods using silica gel plates (60 F254).

3.2 Degradation

products

Degradation products were analysed for in leachates and soil.


for degradation

products

Comparison of radioactive areas with unlabelled standards of parent and

known degradation products.

3.3 Reference substance Parent and known metabolites:

TI-435 (unlabelled parent)

Guanidine hydrochloride

Methylguanidine hydrochloride

NTG: Nitroguanidine


Urea

N-Methylurea






Cyanamide

Cyanamide acid



TZG: N-(2-chloro-5-thiazolylmethyl)-N’-guanidine




for reference

substance

Analysis by standard TLC methods as described in section 3.1.6.

3.4 Soil types Depending on the soil layer, the sand content ranged from 68-82% and

the clay content from 8-19%. In the 0-10 cm layer, the organic C content

was 1.8% and the pH was 6.6 (0.01 M CaCl2). For further information

see Table A7_2_3_2_01-1


3.5.1 Test system Undisturbed soil core: 1 m2 surface area, 1.3 m depth

sandy loam (Laacherhof AXXa)

Removed from the field in April 1994 and installed in an open lysimeter

facility (assignment: lysimeter no. 24).


application

Thiazolyl[methylene-14

C]TI-435 formulated as WS 70

Applied as seed treatment in two consecutive years: equivalent to 100 g

a.s./ha on winter barley in September 1996 and 137.5 g a.s./ha on winter

wheat in October 1997; total actual amount of 23.75 mg a.s. per

lysimeter.

In October 1998 (3rd

year), untreated winter wheat was sown. During the

entire study, the surrounding (6.7 x 3.3 m) was sown uniformly with the

lysimeter.

3.5.3 Test conditions Relevant meteorological data like temperature (air and soil), rainfall, air

humidity, sunshine and wind velocity were recorded in the vicinity of the

lysimeter station (about 1 or 0.5 km apart). The lysimeter was

additionally irrigated in the first (109 mm water) and second year

(216 mm water).

The total precipitation + irrigation of each year were:

year 1 : September 1996 - August 1997 : 877.7 mm



3.6 Sampling and

work-up

3.6.1 Leachates In general, leachates were sampled every month (if they occurred, or

even twice per month in case of high quantities). The volume of each

leachate was measured and the radioactivity determined (Liquid

Scintillation Counting). The samples were deep-frozen at -20°C until

processing. The storage stability of the residues was confirmed over a

period of 24 month. Individual leachate samples were concentrated with

a vacuum evaporator and separated directly on TLC plates. Pooled

leachates of the 3rd

year were worked up by means of Craig partition.

Butanol as mobile phase and water as stationary phase were used to

separate the residues. The partition was repeated 150 times within about

8 hours. After separation in the Craig apparatus, the phases were

homogenised with propanol. Three fractions were finally obtained and






evaluated on TLC silica gel plates using two solvent systems (selectivity

for either polar or non-polar compounds). The radioactive zones on the

TLC plates were measured by means of a Bio-Imaging Analyzer. Total

radioactive residues were expressed as TI-435 equivalents (parent

equiv.).

3.6.2 Soil At the end of the study, the upper three soil layers (10-cm layers, 0-

30 cm) were completely removed from the lysimeter and separately

homogenised in a cement mixing machine. The deeper layers (30-

130 cm) were samples with a corer (three drill cores of about 50 cm2).

Up to the 50 cm layer, aliquots of the soil were extracted (at room

temperature and thereafter hot). Non-extractable residues were

determined by combustion of the extracted soils.

3.6.3 Plants The cereals were harvested in July 1997 (treated barley), August 1998

(treated wheat) and July 1999 (untreated wheat). The stems were cut off

directly above the ground and the ears (grains plus hulls) separated form

the straw. For analysis, the hulls were added to the straw. The samples

were homogenised under liquid nitrogen. Aliquots were combusted to

determine the Total Radioactive Residues (TRR). Grains were extracted

under reflux. The residues in the extracts were separated and identified

on TLC. The top soil layer (0-20 cm) was turned over with a spade after

the first and second harvest.

4 RESULTS

4.1 Material balance At the end of the study, a material balance was determined. The

distribution of the Total Radioactive Residues (TRR) is summarised in

Table A7_2_3_2_01-2. The losses of radioactivity (calculated value)

were attributed to mineralisation.

4.2 Leachates The total of precipitation and irrigation was 2650 mm at the end of the

study. About 36% of this amount could be recovered as leachate (Table

A7_2_3_2_01-3).

The concentration in the leachates was below 0.030 µg parent equiv./L

until March 1997 and increased to 0.052 µg parent equiv./L in May 1997

(first year). About 0.096 µg parent equiv./L were found in the leachate of

January 1998, and thereafter the concentrations varied between 0.069 µg

and 0.033 µg parent equiv./L (second year). The maximum concentration

was determined in the leachate sample of November 1998 and was

0.133 µg parent equiv./L. In the next leachate of December 1998,

0.112 µg parent equiv./L were detected. Thereafter, the concentrations

decreased again until the end of the study (last leachate May 1999:

0.073 µg parent equiv./L).

Additionally, annual leachate samples of the 2nd

and 3rd

year were

prepared by combining individual leachates. The maximum

concentration based on these annual samples was observed in the 3rd

year

and was 0.104 µg parent equiv./L (see Table A7_2_3_2_01-3). In

addition, the three leachates with the highest concentrations (i.e. of

January, November and December 1998) were analysed separately to

identify the residues. The distribution of the radioactivity is provided in

Table A7_2_3_2_01-4.

TI-435 was not detected in any of the leachates analysed. Known

metabolites could not be detected, whereas four unknowns were seen. In

the annual samples, the maximum concentration of a single unknown

compound was 0.065 µg parent equiv./L (U3, 3rd

year).






4.3 Soil The distribution of the applied radioactivity in the soil profile was

determined at the end of the study. The majority of the radioactivity was

determined in the top soil (0-20 cm) and amounted to 51.6% of the

applied amount. In the following two layers, 5.2% (20-30 cm) and 1.2%

(30-40 cm) of the applied radioactivity were determined. Only negligible

amounts were measured in deeper layers (Table A7_2_3_2_01-5).

The characterisation of the radioactivity in the three top layers is

provided in Table A7_2_3_2_01-6.

4.4 Plants The total radioactive residue in the grains of the first crop winter barley

was 20.5 µg parent equivalent/kg. In the second crop, after the second

application of TI-435, the concentration in wheat grains increased to

35.7 µg/kg parent equivalents. A low amount was determined in the

grains of the 3rd

crop wheat (3.9 µg parent equivalent/kg). For details see

Table A7_2_3_2_01-7.

From the total radioactive residues, 18.3% could be extracted from the

barley grains and 34.6% from the wheat grains (2nd

crop). With respect

to the low concentration in the 3rd

crop (wheat), no extraction was

performed. The parent compound and the metabolite TZNG could be

identified in the extracts. The concentration of TI-435 was found to be

0.7 µg/kg in barley grains and 1.1 µg/kg in wheat grains (Table

A7_2_3_2_01-8).


5.1 Materials and

methods

The German BBA guideline for lysimeter studies is an internationally

accepted guideline. No relevant deviations from the guideline occurred.

5.2 Results and

discussion

Distribution of radioactivity

The majority of the applied radioactivity was recovered from the soil

(about 60% of applied) and about 37% of applied was attributed to losses

by mineralisation.

Degradation and residues in soil, leachate and plants

The parent compound and the metabolite TZNG were identified in the

soil samples.

In the total leachates, less than 0.3% of applied radioactivity was

detected. The highest annual concentration was found for the 3rd

year and

was 0.104 µg parent equiv./L. Neither TI-435 nor any known metabolites

were identified in the leachates analysed. Unknown metabolite U3

represented the highest individual concentration (0.065 µg parent

equiv./L) in the annual leachate of the 3rd

year.

In the crops, 3.2% of the applied radioactivity was measured. The total

radioactive residue in the grains was highest in the 2nd

crop

(35.7 µg parent equiv./kg in winter wheat). TI-435 (maximum:

1.1 µg/kg) and the metabolite TZNG (maximum: 4.2 µg parent

equiv./kg) were identified.

5.3 Conclusion On the basis of these findings, TI-435 and its degradation products are

not expected to occur in relevant amounts in groundwater. Degradation

in soil continuous. No accumulation in plants is to be expected.

5.3.1 Reliability 1









Date 2004/11/19



Conclusion TI-435 and its degradation products are not expected to occur in relevant amounts

in groundwater. No accumulation in plants is to be expected.

Reliability 1


Remarks

COMMENTS FROM ...

Date



Conclusion

Reliability

Acceptability

Remarks



Table A7_2_3_2_01-1: Soil characteristics

Classification Sandy loam

Depth [cm)] 0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130

sand [%]

silt [%]

clay [%]

70.8

20.7

8.6

69.4

22.2

8.5

68.3

21.1

10.6

67.5

20.7

11.8

70.3

18.0

11.8

71.7

15.2

12.6

75.5

15.1

9.4

73.2

13.0

13.8

68.7

12.8

18.5

70.2

12.2

17.6

78.0

10.4

11.6

77.3

8.8

13.8

815

10.2

8.3

organic C [%] 1.8 1.1 0.7 0.5 0.3 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1

pH (water) 7.2 7.2 7.2 7.3 7.5 7.5 7.6 7.7 7.7 7.7 7.7 7.7 7.7

pH (CaCl2) 6.6 6.4 6.3 6.5 6.5 6.5 6.5 6.5 6.6 6.6 6.6 6.6 6.7

Table A7_2_3_2_01-2: Balance of Total Radioactive Residues (100% = 107.14 MBq resulting from two

applications)

Leachates Crops Soil Losses TOTAL

grains straw + hulls (mineralisation)

0.28% 0.25% 2.92% 59.29% 37.26%* 100%

* calculated value

Table A7_2_3_2_01-3: Annual amounts of leachates and total radioactive residues

Amount Total radioactive residues

[L] [µg parent equivalent/L]

1st year (Sept. 1996 to Aug. 1997) 273 0.013*

2nd

year (Sept. 1997 to Aug. 1998) 190 0.062

3rd

year (Sept. 1998 to Aug. 1999) 494 0.104

Sum : 956 Average: 0.060

Mean per year : 319

* calculated based on monthly samples

Table A7_2_3_2_01-4: Distribution of radioactivity in leachates

Sample Total charact. Origin TI-435 U3 U4 U5 U7 Diffuse

date [µg parent equivalent/ L]

3rd

year* 0.093 N.D. N.D. 0.065 0.012 0.001 0.003 0.012

Jan. 1998 0.096 n.d. n.d. 0.059 0.020 n.d. n.d. 0.017

Nov. 1998 0.133 n.d. n.d. 0.072 0.014 n.d. n.d. 0.047

Dec. 1998 0.112 n.d. n.d. 0.069 0.027 n.d. n.d. 0.015

* Quantification was performed by means of Craig partition. Limit of detection was about 0.001 µg/L (N.D.).

n.d. not detected (<0.02 µg/L)



Table A7_2_3_2_01-5: Distribution of radioactivity in the soil profile (sampling at the end of the study)

Soil layer Total radioactive residue Radioactivity

[cm] [µg parent equivalent/kg soil] [% of applied]

0-10 55.5 28.0

10-20 47.0 23.6

20-30 9.6 5.2

30-40 2.0 1.2

40-50 0.76 0.5

50-60 0.31 0.2

60-70 0.23 0.2

70-80 0.15 0.1

80-90 0.14 0.1

90-100 0.07 0.0

100-110 0.06 0.0

110-120 0.07 0.1

120-130 0.06 0.0

TOTAL - 59.3

Table A7_2_3_2_01-6: Distribution of radioactivity in soil extracts (sum of cold and hot extract)

Soil layer Origin TI-435 TZNG TMG U5 Diffuse

[cm] [µg/kg]*

0 - 10 33.4 1.23 25.4 5.21 n.d. n.d. 1.19

10 - 20 31.0 1.05 24.7 3.76 n.d. n.d. 1.25

20 - 30 6.69 0.32 5.48 0.55 n.d. n.d. 0.31

Total 70.84 2.59 55.5 9.49 n.d. n.d. 2.75

n.d. not detected (<0.01 µg/kg)

* Not identified zones (origin, U5 and diffuse radioactivity) were calculated as TI-435 equivalents. TZNG

and TMG were calculated based on their molecular weight.

Table A7_2_3_2_01-7: Yields and total radioactive residues (TRR) in crops

Plant 1st year 2

nd year 3

rd year

part winter barley* winter wheat* winter wheat

yield

[g/m2]

TRR

[µg par. equiv./kg]

yield

[g/m2]

TRR

[µg par.equiv./kg]

yield

[g/m2]

TRR

[µg par.equiv./kg]

Grain 1250 20.5 849 35.7 980 3.9

Straw & hulls 1969 126 840 353 829 60

* Crops grown from treated seeds



Table A7_2_3_2_01-8 Distribution of radioactivity in grains of 1st and 2

nd crop

Extracted Not extracted Origin TI-435 TZNG Diffuse

Plant part [µg parent equiv./kg]

barley grains/ 1st year 3.75 16.7 1.8 0.7 1.0 0.1

wheat grains/ 2nd

year 12.35 23.3 5.5 1.1 4.2 1.4

* TZNG was calculated based on molecular weight







use only

1.1 Reference Stupp, H.P. (2001c): Degradation and translocation behavior of the

insecticide TI-435 in a lysimeter under field conditions.

Bayer AG, 51368 Leverkusen, Germany; unpublished report no.

MR599/00


1.2.1 Data owner Sumitomo Chemical Takeda Agro Co., Ltd. and Bayer CropScience


letter of access

No


protection




Germany BBA Part IV, 4-3

2.2 GLP Yes

2.3 Deviations No major deviations



C]TI-435

3.1.1 Radiolabelling [Nitroimino-14

C]TI-435


3.1.3 Specific radioactivity 3.78 MBq/mg

3.1.4 Purity Radiochemical purity: > 99% according to radio TLC

Chemical purity: > 99% according to HPLC


properties

TS not inhibitory to microorganisms

TS not volatile (vapour pressure: 1.3 · 10-10

Pa)

Water solubility (20°C): 327 mg/L (see Morrissey & Kramer, 2000a)

3.1.6 Method of analysis Analysis by TLC methods using silica gel plates (60 F254).

3.2 Degradation

products

Degradation products were analysed for in leachates and soil.


for degradation

products

Comparison of radioactive areas with unlabelled standards of parent and

known degradation products.

3.3 Reference substance Parent and known metabolites:

TI-435 (unlabelled parent)

Guanidine hydrochloride

Methylguanidine hydrochloride

NTG: Nitroguanidine


Urea

N-Methylurea

Cyanamide






Cyanamide acid



TZG: N-(2-chloro-5-thiazolylmethyl)-N’-guanidine




for reference

substance

Analysis by standard TLC methods as described in section 3.1.6.

3.4 Soil types Depending on the soil layer, the sand content ranged from 68-82% and

the clay content from 8-19%. In the 0-10 cm layer, the organic C content

was 1.8% and the pH was 6.6 (0.01 M CaCl2). For further information

see Table A7_2_3_2_02-1


3.5.1 Test system Undisturbed soil core: 1 m2 surface area, 1.3 m depth

sandy loam (Laacherhof AXXa)

Removed from the field in April 1996 and installed in an open lysimeter

facility (assignment: lysimeter no. 17 and 18).


application

[Nitroimino-14

C]TI-435 was formulated as SC 200

Applied as spray in two consecutive years: equivalent to 160 g a.s./ha on

grass in July 1997 and May 1998; total actual amount of 31.91 mg a.s.

on lysimeter no. 17 and 32.49 mg a.s. on lysimeter no. 18

The grass on the lysimeters and surrounding (about 3x3 m around each

lysimeter) was sown in June 1997.

3.5.3 Test conditions Relevant meteorological data like temperature (air and soil), rainfall, air

humidity, sunshine and wind velocity were recorded in the vicinity of the

lysimeter station (about 1 or 0.5 km apart). The lysimeters were

additionally irrigated in the first (168 mm water), second (45 mm water)

and third year (150 mm water).

The total precipitation + irrigation of each year were:

year 1 : July 1997 - July 1998 : 846.8 mm

year 2 : July 1998 - July 1999 : 929.6 mm

year 3 : July 1999 - July 2000 : 851.9 mm

3.6 Sampling and

work-up

3.6.1 Leachates In general, leachates were sampled every month (if they occurred). The

volume of each leachate was measured and the radioactivity determined

(Liquid Scintillation Counting). The samples were deep-frozen at -20°C

until processing. The storage stability of the residues was confirmed over

a period of 18 month.

Individual leachate samples, which were taken at the maximum Total

Radioactive Residue (TRR) and at intervals of about half a year, were

analysed for the parent and degradation products. The samples were

concentrated with a vacuum evaporator and separated directly on TLC

plates. Additionally, annual leachate samples were combined from

individual samples with respect to the amount of leachate. They were

worked up by means of Craig partition (for description of method see

Stupp, 2001b). The radioactive zones on the TLC plates were measured

by means of a Bio-Imaging Analyzer. Total radioactive residues were






expressed as TI-435 equivalents (parent equiv.).

3.6.2 Soil At the end of the study (August 2000), the upper three soil layers (10-cm

layers, 0-30 cm) were completely removed from the lysimeter and

separately homogenised in a cement mixing machine. The deeper layers

(30-130 cm) were sampled with a corer (three drill cores per lysimeter

with about 50 cm2). Up to the 50 cm layer, aliquots of the soil were

extracted with acetonitrile and water (at room temperature and thereafter

hot). Non-extractable residues were determined by combustion of the

extracted soils.

3.6.3 Plants Grass was regularly cut and the cut remained on the lysimeters as mulch

and rotted. The grass plants were mixed into the upper soil layer

(0-10 cm) before further analysis. Therefore, grass cut and remaining

grass plants were not analysed separately.

4 RESULTS

4.1 Material balance At the end of the study, a material balance was determined. The

distribution of the Total Radioactive Residues (TRR) is summarised in

Table A7_2_3_2_02-2. The losses of radioactivity (calculated values)

were attributed to mineralisation.

4.2 Leachates The total of precipitation and irrigation was 2628 mm at the end of the

study. About 49% of this amount could be recovered as leachate. The

two lysimeters behaved very similarly (Table A7_2_3_2_02-3).

The concentrations in the leachates were below 0.030 µg parent equiv./L

until January/February 1998 and increased to 0.196 µg parent equiv./L

(Lysimeter 17) respective 0.275 µg parent equiv./L (Lysimeter 18) in

June/July 1998 (first year). During autumn, the concentrations

approximately remained on this level before reaching in

November/December the peak concentrations of 0.402 µg (Lysimeter

17) and 0.514 µg parent equiv./L (Lysimeter 18). Thereafter, the

concentrations decreased (until December 1999) before increasing again

to 0.354 µg (Lysimeter 17) and 0.434 µg parent equiv./L (Lysimeter 18)

in March 2000.

Additionally, annual leachate samples were prepared by combining

individual leachates. The maximum concentration based on these annual

samples was observed in the 2nd

year and was 0.349 µg and 0.417 µg

parent equiv./L (see Table A7_2_3_2_02-3). The mean of the three years

and two lysimeters was calculated to be 0.281 µg parent equiv./L.

TI-435 was not detected in any individual leachates. MNG and NTG

amounted to maximum 0.116 µg/L (Lysimeter 18) and 0.074 µg/L

(Lysimeter 18), respectively. The distribution of the radioactivity in the

annual leachates is provided in Table A7_2_3_2_02-4.

Beside MNG and NTG, six unknown degradation products were seen.

The maximum concentrations of MNG and NTG occurred in the 3rd

year

and were, calculated as mean of the two lysimeters, 0.066 µg/L (MNG)

and 0.031 µg/L (NTG).






4.3 Soil The distribution of the applied radioactivity in the soil profile was

determined at the end of the study. The major part of the radioactivity

was determined in the top 10 cm and amounted to 30.4% of the applied

amount. In the next layer (10-20 cm), 7.3% of the applied radioactivity

were determined. Only negligible amounts were measured in deeper

layers (Table A7_2_3_2_02-5).

The characterisation of the radioactivity in the three top layers is

provided in Table A7_2_3_2_02-6.


5.1 Materials and

methods

The German BBA guideline for lysimeter studies is an internationally

accepted guideline. No relevant deviations from the guideline occurred.

5.2 Results and

discussion

5.3 Conclusion On the basis of these findings, TI-435 and its degradation products are

not expected to occur in relevant amounts in groundwater. Degradation

in soil continuous. No accumulation in plants is to be expected.

5.3.1 Reliability 1









Date 2004/11/19



Conclusion TI-435 and its degradation products are not expected to occur in relevant amounts

in groundwater. No accumulation in plants is to be expected.

Reliability 1


Remarks

COMMENTS FROM ...

Date



Conclusion

Reliability

Acceptability

Remarks



Section A7.3.1 Annex Point IIIA VII.5

Phototransformation in air (estimation method)

1 REFERENCE

Official

use only

1.1 Reference Hellpointner, E. (1998): Calculation of the chemical lifetime of TI-435

in the troposphere.


705/98




letter of access

None


protection



2.1 Guideline study No

No guideline specified; an estimation was made according to:

Atkinson, R. (1985): Kinetics and mechanisms of the gas-phase:

Reactions of the hydroxyl radical with organic compounds under

atmospheric conditions. Chem. Rev., 86, 69-201.

Atkinson, R. (1988): Estimation of gas-phase hydroxyl radical rate

constants for organic chemicals. Environ. Toxicol. Chem., 7, 435-442.

X

2.2 GLP No (not relevant)

2.3 Deviations Not relevant


3.1 Substance TI-435

3.2 Model calculation The computer program AOPWIN version 1.87 (8/98) was used. X

4 RESULTS

4.1 Photochemical

reactions

Based on the molecular structure of TI-435, mainly reactions with

photochemically produced hydroxyl radicals are expected to determine

the degradation rate and chemical lifetime of TI-435 in the air.

According to ATKINSON, the total reactivity is divided in 4 single

contributions of which reaction with nitrogen, hydrogen abstractions and

addition of hydrogen to the aromatic ring are relevant for TI-435.

4.2 Half-life in air Based on this overall OH rate constant of 136.97 x 10-12

cm3/molecule

sec., a half-life of 1 hour for TI-435 in air is assumed. This corresponds

to a chemical lifetime of 1.4 hours using a 12-hour day which is regarded

as the global 12-hour day time concentration (excluding the night).

This estimation does not consider any contribution of an attack by other

radicals (i.e. by nitrate radicals). Furthermore, it should be taken into

account that the OH radical concentration in the night decreases to zero.

A more conservative assessment of the overall OH radical rate constant

(e.g. not considering the addition of OH radicals to the aromatic ring)

would result in a maximum chemical lifetime of 1.5 hours in air.

X






5.1 Materials and

methods

The chemical lifetime of TI-435 in the troposphere was calculated using

the computer program AOPWIN version 1.87 (8/98).

5.2 Results and

discussion

A half-life of 1 hour for TI-435 in air is assumed corresponding to a

chemical lifetime of 1.4 hours using a 12-hour day which is regarded as

the global 12-hour day time concentration (excluding the night).

X

5.3 Conclusion Based on the relatively short chemical lifetime of TI-435, accumulation

in the air is not to be expected.

X

5.3.1 Reliability 1




Date 2006-05-19

Guideline and Quality

Assurance

Applicant’s version is acceptable.

Comment:

The reference Atkinson, R. (1985) is published in Chem. Rev. volume 85 and not

in volume 86.

Materials and Methods In general, applicant’s version is acceptable.

Comments:

At first, applicant used the following model input parameter for the estimation of

the phototransformation of the a.s. in the air (see Hellpointer, 1998):

12-hours-day with a OH radical concentration of 1.5x106 radicals cm

-3 (= global

12-hours-day-time concentration excluding the night).

However, these input parameters are not in agreement with the input parameters

fixed in the EU TGD on Risk Assessment (2003), Part II, Chapter 2.3.6.3:


-3 (=global

24-hours mean concentration).

The applicant was requested by the C.A. to submit a further estimation of the

phototransformation in air using the EU standard input parameters. This second

estimation is available under Doc. IV-A, Section No. 7.3.1. In this estimation

the revised version 1.91 of the software programme AOPWIN was applied.





Results and discussion In general, applicant’s version is acceptable.

The chemical lifetime was introduced by the applicant. The definition of the

chemical lifetime is as follows:

2ln

life_halflifetime_chemical

Comments:

The applicant’s conservative assessment of the chemical lifetime of the a.s.

resulting in a chemical lifetime of 1.5 hours is not transparently documented.

The overall OH rate constant consists of the contributions “hydrogen

abstraction”, “reaction rates with nitrogen, sulfur and –OH” and “OH addition

to aromatic rings”. In the original study, p.3, “results” as well as in the study

summary, item 4.2, “half-life in air” the applicant stated that for the

conservative assessment the contribution of the assumed “OH addition to

aromatic rings” was not taken into account (marked with ** in appendix 4 of

the original study). In contrast, in the original study, p. 3, “summary” the

applicant stated that the conservative assessment is solely based on the

contribution of OH radical reaction (= “reaction rates with nitrogen, sulfur

and –OH”).

Calculations of C.A. show

- a chemical lifetime of 1.5 hours using the contribution of the OH radical

reaction (= not taken into account the assumed “OH addition to aromatic

rings”and the “hydrogen abstraction”) and

- a chemical lifetime of 1.4 hours using the contribution of the “hydrogen

abstraction” and the “reaction rates with nitrogen, sulfur and –OH” (= not

taken into account the assumed “OH addition to aromatic rings”).

The evaluated study summary comprises only the results of the primarily

submitted original study. Results of the second original study submitted later

in the dossier evaluation process are only considered by the C.A. in the

conclusion (see below) and in Doc. II-A, Environmental Effect Assessment.

As the results of both original studies do not affect the overall conclusion for

the behaviour of the a.s. in air a revised version of the study summary

(including the results of the later handed out original study) was not requested

by C.A.

Conclusion The halflife of the a.s. in the troposphere was calculated using the software

programme AOPWIN. A halflife of 0.94 hours for the a.s. in air is estimated

corresponding to a chemical lifetime of 1.4 hours using a 12-hours-day with a OH

radical concentration of 1.5x106 radicals cm

-3 which is regarded as the global 12-

hours-day-time concentration (excluding the night). A halflife of 2.81 hours for

the a.s. in air is estimated corresponding to a chemical lifetime of 4.1 hours using a


-3 which is

regarded as the global 24-hours-mean concentration.

Based on these results, an accumulation of the a.s. in the air is not to be expected.

Reliability 1

Acceptability The two original studies (Doc. IV-A, Section No. 7.3.1) as well as the study

summary (Doc. III-A, Section No. 7.3.1) are acceptable, although there are some

minor deficiencies in the study summary.

Remarks

COMMENTS FROM ...













Remarks



Section A7.4.1.1 Annex Point IIA7.1

Acute toxicity to fish

1 REFERENCE

Officialuse only



1.2.1 Data owner




2.1 Guideline study Yes OECD No. 203 (1992) in compliance with the Directive 92/69/EEC C.1

2.2 GLP Yes

2.3 Deviations No


3.1 Test material


3.1.2 Specification

3.1.3 Purity


Not applicable



3.2 Preparation of TS solution for poorly soluble or volatile test substances


No


3.4.1 Dilution water





3.4.2 Test organisms

3.4.3 Test system


3.4.5 Duration of the test 96 h

3.4.6 Test parameter Mortality

3.4.7 Sampling

3.4.8 Monitoring of TS concentration

3.4.9 Statistics

4 RESULTS

4.1 Limit Test

4.1.1 Concentration

4.1.2 Number/ percentage of animals showing adverse effects

4.1.3 Nature of adverse effects

4.2 Results test substance

4.2.1 Initial concentrations of test substance

X

4.2.2 Actual concentrations of test substance

4.2.3 Effect data (Mortality)

4.2.4 Concentration / response curve

4.2.5 Other effects

4.3 Results of controls

4.3.1 Number/ percentage of animals showing adverse effects


4.4 Test with reference substance







The acute toxicity of TI-435 Technical to fish (rainbow trout) was determined in a static limit test according to OECD No. 203 (1992) in compliance with the Directive 92/69/EEC C.1. The nominal test concentration was 100 mg a.s./L.


None of the seven test animals died during exposure for 96 h and none of the control animals died within this time.

5.2.1 LC0 100 mg/L

5.2.2 LC50 > 100 mg/L

5.2.3 LC100 > 100 mg/L

5.3 Conclusion The validity criteria can be considered as fulfilled.

5.3.1 Reliability 1





Date 2004-10-04



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks



Section A7.4.1.2/01 Annex Point IIA7.2

Acute toxicity to invertebrates Daphnia magna

1 REFERENCE

Officialuse only

1.1 Reference (2000b):


1.2.1 Data owner




2.1 Guideline study Yes OECD No. 202 (1984) and Directive 92/69/EEC C.2

2.2 GLP Yes

2.3 Deviations No (to EC Directive)


3.1 Test material


3.1.2 Specification

3.1.3 Purity









3.4.3 Test system







3.4.6 Test parameter Mortality and immobility

3.4.7 Sampling Sampling intervals: 0 and 48 h


3.4.9 Statistics

4 RESULTS

4.1 Limit Test Not performed



Nominal test concentrations: 0, 7.5, 15, 30, 60 and 120 mg a.s./L


4.2.3 Effect data (Immobilisation)


4.2.5 Other effects





The acute toxicity of TI-435 Technical to invertebrates (Daphnia magna) was determined in a static test according to OECD No. 202 (1984) in compliance with the Directive 92/69/EEC C.2. Duplicate test solutions with the following nominal test concentrations were used: 0, 7.5, 15, 30, 60 and 120 mg a.s./L.


None of the daphnids in the test and control solutions showed any adverse effects.

5.2.1 EC0 120 mg a.s./L X

5.2.2 EC50 > 120 mg a.s./L X

5.2.3 EC100 > 120 mg a.s./L X


5.3.1 Reliability 1









Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks





1 REFERENCE

Officialuse only



1.2.1 Data owner




2.1 Guideline study Yes OECD No. 202 (1984) and Directive 92/69/EEC C.2

2.2 GLP Yes

2.3 Deviations No


3.1 Test material


3.1.2 Specification

3.1.3 Purity


Not applicable








3.4.3 Test system






3.4.5 Duration of the test 48 hours

3.4.6 Test parameter Immobility (determined after 24 and 48 hours)

3.4.7 Sampling


3.4.9 Statistics

4 RESULTS

4.1 Limit Test



Nominal test concentrations: 0, 1.0, 3.2, 10, 32, 100 and 270 mg/L


4.2.3 Effect data (Immobilisation)


4.2.5 Other effects



4.4.1 Concentrations

4.4.2 Results



The acute immobilisation toxicity of TI-435 Technical to invertebrates (Daphnia magna STRAUS) was determined in a static test according to OECD No. 202 (1984) and Directive 92/69/EEC C.2. The following nominal test concentrations were used: 0, 1.0, 3.2, 10, 32, 100 and 270 mg/L. No deviations to the test guidelines occurred.






After 24 hours of exposure to TI-435 Technical, no immobility of daphnids was observed up to the highest test concentration. However, at the end of the test (48 hours), immobile daphnids were observed at test concentrations ≥ 10 mg/L.

5.2.1 EC0 (48 hours) 3.2 mg/L

5.2.2 EC50 (48 hours) 40 mg/L (confidence interval: 31 – 51 mg/L) X

5.2.3 EC100 (48 hours) > 270 mg/L


5.3.1 Reliability 1





Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks




Growth inhibition test on algae

1 REFERENCE

Officialuse only



1.2.1 Data owner





2.2 GLP Yes

2.3 Deviations

X


3.1 Test material


3.1.2 Specification

3.1.3 Purity







3.4.1 Culture medium




Growth inhibition test on algae


3.4.3 Test system



3.4.6 Test parameter Cell density, biomass (area under the growth curve) and growth rate

3.4.7 Sampling


3.4.9 Statistics

4 RESULTS

4.1 Limit Test



Nominal concentrations: 0, 3.8, 7.5, 15, 30, 60 and 120 mg a.s./L


4.2.3 Growth curves


4.2.5 Cell concentration data



X





The toxicity of TI-435 towards the freshwater alga Selenastrum capricornutum was tested according to FIFRA Subdivision J Series 123-2 which is in compliance with EC Directive 92/69/EEC, C.3. A static test over 120 hours with nominal concentrations of 0, 3.8, 7.5, 15, 30, 60 and 120 mg a.s./L was performed.


The EC50-value for cell density was calculated to be 75 mg a.s./L after 72 h. The values for biomass and growth are given below.

X

5.2.1 NOErC 15 mg a.s./L (72 h) X

5.2.2 ErC50 > 120 mg a.s./L (72 h) X

5.2.3 EbC50 70 mg a.s./L (72 h) X


5.3.1 Reliability 1







Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks

X





The toxicity of TI-435 towards the freshwater alga Selenastrum capricornutum was tested according to FIFRA Subdivision J Series 123-2 which is in compliance with EC Directive 92/69/EEC, C.3. A static test over 120 hours with nominal concentrations of 0, 3.8, 7.5, 15, 30, 60 and 120 mg a.s./L was performed.


The EC50-value for cell density was calculated to be 75 mg a.s./L after 72 h, 52 mg/L after 96 h and 57 mg/L after 120 h. The values for biomass and growth are given below.

X

6.2.1 NOErC 15 mg a.s./L (72 h, 96 h), 30 mg/L (120 h) X

6.2.2 ErC50 > 120 mg a.s./L (72 h, 96 h, 120 h) X

6.2.3 EbC50 70 mg a.s./L (72 h), 55 mg/L (96 h), 56 mg/L (120 h) X


6.3.1 Reliability 1





Inhibition to microbial activity (aquatic)


use only

1.1 Reference Bealing, D.J. and Watson, S. (2000): TI-435 technical: Determination of

inhibition of respiration of activated sludge.

Covance Laboratories Ltd, Harrogate, North Yorkshire HG3 1PY,

England; unpublished report 586/210-D2145




letter of access

None


protection




Directive 88/302/EEC, Part C, identical to OECD 209

2.2 GLP Yes

2.3 Deviations No


3.1 Test material As given in section 2

3.1.1 Lot/Batch number 50071820 (see Certificate of Analysis in Appendix 1 in the report)

3.1.2 Specification As given in section 2

3.1.3 Purity

3.1.4 Composition of

Product

Not applicable


properties



3.1.6 Method of analysis No analysis of test substance performed

3.2 Preparation of TS

solution for poorly

soluble or volatile

test substances

See Table A7_4_1_4-1

3.3 Reference

substance

Yes

3,5-dichlorophenol


for reference

substance

No analysis of reference substance performed


3.4.1 Culture medium Synthetic sewage concentrate (according to OECD 209, 100-fold)

3.4.2 Inoculum /

test organism

See Table A7_4_1_4-2

3.4.3 Test system See table A7_4_1_4-3

3.4.4 Test conditions See table A7_4_1_4-4






3.4.6 Test parameter Respiration inhibition

3.4.7 Analytical

parameter

Oxygen measurement

3.4.8 Sampling at the end of the study after 3 hours

3.4.9 Monitoring of TS

concentration

no

3.4.10 Controls 4 controls without test substance

3.4.11 Statistics

4 RESULTS


4.2 Results test

substance

4.2.1 Concentrations of

test substance

1.0, 10, 100, and 1000 mg/L

4.2.2 Concentration/

response curve

4.2.3 Effect data The treatment with TI-435 Technical did not result in significant

inhibition at any of the concentrations (no reduction of the respiration

rate compared to the controls). Accordingly, the EC50 for TI-435

Technical is > 1000 mg/L.


4.4 Test with

reference

substance


4.4.2 Results


5.1 Materials and

methods

TI-435 Technical was tested for its inhibition to microbial activity

according to Directive 88/302/EEC, Part C (identical to OECD 209).

Activated sludge was exposed to 1.0, 10, 100, and 1000 mg a.s./L and

the respiration rate measured in comparison to control solutions and to

the reference substance 3,5-dichlorophenol.

5.2 Results and

discussion

In a 3-hour range-finding test, TI-435 Technical did not inhibit the

respiration of activate sludge at concentrations up to and including

1000 mg/L.

5.2.1 EC50 > 1000 mg/L (nominal)





5.3 Conclusion The control respiration rates used to obtain the mean lay within 15% of

each other. The EC50 of the reference inhibitor, 3,5-dichlorophenol, lay

between 5 and 30 mg/L, indicating that the sludge used in this study was

neither unusually sensitive nor abnormally robust. Thus, the validity

criteria can be considered as fulfilled.

5.3.1 Reliability 1






Date 2005-08-17

Materials and Methods applicants version is acceptable

Results and discussion applicant's version is adopted


Reliability 1


Remarks none

COMMENTS FROM ...









Remarks



Table A7_4_1_4-1: Preparation of TS solution for poorly soluble or volatile test substances

Criteria Details

Dispersion Yes

Vehicle No

Procedure Due to insufficient solubility, the required exposure

concentrations were achieved by making direct

additions of the test substance to the test vessels

which contained reverse-osmosis water and synthetic

sewage. The test substance was homogeneously

dispersed by ultrasonication before adding the

inoculum.

Table A7_4_1_4-2: Inoculum / Test organism

Criteria Details

Nature Activated sludge

Source Sewage treatment plant treating predominantly

domestic sewage

Sampling site Burley Menston sewage treatment works (Yorkshire

Water)

Laboratory culture No

Preparation of inoculum for exposure Dilution to a nominal solids content of 4 g/L, pH

adaptation from 5.91 to 6.51 (with 1 M NaOH)

Pretreatment Aeration and feeding overnight with synthetic sewage

on two occasions

Initial cell concentration 4 g suspended solids/L

Table A7_4_1_4-3: Test system

Criteria Details

Culturing apparatus Culture flasks

Number of culture flasks/concentration 2

Aeration device Compressed air was delivered to each test vessel by a

Pasteur pipette.

Measuring equipment Dissolved oxygen (DO) was measured with a DO

probe.

Test performed in closed vessels due to significant

volatility of TS

No



Table A7_4_1_4-4: Test conditions

Criteria Details

Test temperature 20.9 to 21.6°C

pH of diluted inoculum 6.51 after adjustment

Aeration of dilution water No

Suspended solids concentration 4 g/L



Bioconcentration





-




Date


Conclusion

Remarks






Remarks



Section A7.4.3.1 Annex Point AIII XIII.2.1

Prolonged toxicity to an appropriate species of fish





-




Date


Conclusion

Remarks






Remarks




Section A7.4.3.2 Annex Point IIIA XIII 2.2

Effects on reproduction and growth rate on an appropriate species of fish

1 REFERENCE

Officialuse only



1.2.1 Data owner




2.1 Guideline study Yes US EPA OPPTS draft guideline No. 850.1400 (1996), US EPA-FIFRA Subdivision E, Series 72-4 (1982) and ASTM Standard E1241-88 (1988)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material


3.1.2 Specification 2

3.1.3 Purity








3.4.3 Handling of embryos and larvae (OECD 210/212)

3.4.4 Test system






3.4.6 Duration of the test 33 days, including a 5-day embryo hatching period and a 28-day post hatch juvenile growth period.

3.4.7 Test substance concentrations

Nominal test concentrations: 1.3, 2.5, 5.0, 10 and 20 mg a.s./L

3.4.8 Preparation of test solutions

3.4.9 Controls X

3.4.10 Test parameters

3.4.11 Examination/ Sampling


3.4.13 Monitoring of other parameters

3.4.14 Statistics

4 RESULTS

4.1 Range finding test


4.3 Hatching

4.3.1 Hatching time





4.3.2 Hatching success

4.4 Mortality

4.5 Growth



The early life-stage toxicity of TI-435 Technical to fish (fathead minnow) was tested according to US EPA OPPTS draft guideline No. 850.1400 (1996), US EPA-FIFRA Subdivision E, Series 72-4 (1982) and ASTM Standard E1241-88 (1988). The guidelines are in compliance with OECD 210. Fathead minnows were exposed 28 days post-hatch to nominal concentrations of 0, 1.3, 2.5, 5.0, 10 and 20 mg a.s./L. Hatching, mortality and growth of the fish was observed.


Exposure to TI-435 at the concentrations tested showed no statistically significant effects on hatching success, larval survival, total length, wet weight or dry weight as compared to the controls.

5.2.1 NOEC 20 mg a.s./L (nominal)

5.2.2 LOEC > 20 mg a.s./L (nominal)

5.2.3 MATC > 20 mg a.s./L (nominal)

5.3 Conclusion

5.3.1 Reliability 1





Date







Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks


Section A7.4.3.3.1 Annex Point IIIA XIII.2.3

Bioaccumulation in an appropriate species of fish





-




Date


Conclusion

Remarks






Remarks




Bioaccumulation in an appropriate invertebrate species


Other existing data [ ] Technically not feasible [ ] Scientifically unjustified [ ]

Limited exposure [ X ] Other justification [ ]



-




Date


Conclusion

Remarks






Remarks

Page 1 of 1




Effects on reproduction and growth rate with an appropriate invertebrate species

1 REFERENCE

Officialuse only



1.2.1 Data owner




2.1 Guideline study Yes OECD No. 211

2.2 GLP Yes

2.3 Deviations None


3.1 Test material


3.1.2 Specification

3.1.3 Purity





3.3.1 Reference substance concentrations








3.4.3 Handling of offspring

3.4.4 Test system


3.4.6 Duration of the test 21 d


Nominal test concentrations: 0.041, 0.12, 0.37, 1.1, 3.3, 9.9 and 29.7 mg/L


3.4.9 Controls

3.4.10 Test parameter

3.4.11 Examination/ sampling


3.4.13 Statistics

4 RESULTS

4.1 Range finding test

4.2 TS concentrations during the test

4.3 Mortality

4.4 Number of young Daphnia

4.5 First appearance of juveniles

4.6 Test of the reference substance







The toxicity of TI-435 Technical to Daphnia magna reproduction was tested according to OECD No. 211 . No deviations to the guideline occurred. Daphnia magna

were exposed in a semi-static test to nominal concentrations of 0.041, 0.12, 0.37, 1.1, 3.3, 9.9 and 29.7 mg/L for 21 days.


5.2.1 NOEC (reproduction)

0.12 mg/L (nominal)

5.2.2 LOEC (reproduction)

0.37 mg/L (nominal)

5.2.3 EC50 (reproduction)

5.7 mg/L

5.2.4 NOEC (mortality of parental)

0.37 mg/L (nominal)

5.2.5 LOEC (mortality of parental)

1.1 mg/L (nominal)

5.2.6 EC50 (mortality of parental)

29.7 mg/L (nominal)

5.3 Conclusion Mortality of female Daphnia did not exceed 20% at the end of the test. The mean number of living offspring produced per parent animal surviving at the end of the test was > 60 in the control groups. The validity criteria can be considered as fulfilled

5.3.1 Reliability 1





Date



Conclusion

Reliability

Acceptability

Remarks





COMMENTS FROM ...







Remarks



Section A7.4.3.5.1/01 Annex Point IIIA XIII 3.4

Effects on sediment dwelling organisms

1 REFERENCE

Official use only



1.2.1 Data owner





Proposal for a BBA guideline "Effects of plant protection products on the development of sediment-dwelling larvae of Chironomus riparius in a water-sediment system." (1995)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material


3.1.2 Specification

3.1.3 Purity





3.3.1 Test water

3.3.2 Test sediment






3.3.4 Test system




Nominal concentrations: 0.1, 0.32, 0.56, 1.0, 1.8, 3.2 and 10 µg/L

3.3.8 Preparation of test solutions and application

3.3.9 Controls




3.3.13 Statistics The EC-values were calculated by Probit analysis. The χ2-test was performed to establish different sensitivities of sexes (p=0.05).

4 RESULTS


4.2 Physical and chemical parameters

4.3 Emergence rate





4.4 Development rate



.


5.2.1 EC15/NOEC (emergence rate)

0.00072 mg a.s./L

5.2.2 EC5 (emergence rate)

0.00057 mg a.s./L


0.00065 mg a.s./L


0.00106 mg a.s./L

5.3 Conclusion Guideline requirements were fulfilled since 90% of the inserted larvae matured to adults in the controls.

5.3.1 Reliability 1







Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks





1 REFERENCE

Officialuse only



1.2.1 Data owner





2.2 GLP Yes

2.3 Deviations No


3.1 Test material


3.1.2 Specification Not applicable

3.1.3 Purity

3.1.4 Appearance




3.3.1 Test water

3.3.2 Test sediment






3.3.4 Test system




A single nominal concentration of 0.1 mg /L was tested (limit test).


3.3.9 Controls




3.3.13 Statistics

4 RESULTS



The physical and chemical parameters were similar between the treatment and the control throughout the study.

4.3 Emergence rate





4.4 Development rate



In a static test, first instars larvae were exposed to a single nominal concentration of 0.1 mg /L for 28 days and emergence and development of midges was observed.


5.2.1 EC15/NOEC (emergence rate and development rate)

≥ 0.1 mg /L

5.3 Conclusion Guideline requirements were fulfilled since 100% of the inserted larvae matured to adults in the controls.

5.3.1 Reliability 1







Date 2004-10-11



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks

Sumitomo Chemical TI-435 April 2010

Harlan Laboratories project no. 852224 Page 1 of 8



1 REFERENCE

Official use only



1.2.1 Data owner




2.1 Guideline study

2.2 GLP Yes

2.3 Deviations


3.1 Test material TI-435


3.1.2 Specification

3.1.3 Purity









3.3.1 Test water

3.3.2 Test sediment


3.3.4 Test system




TI-435 technical: 0.007, 0.013, 0.025, 0.050 and 0.10 mg/L


3.3.9 Controls




3.3.13 Statistics





4 RESULTS



4.3 Immobilisation







The acute toxicity of TI-435 to larvae of Chironomus riparius was

determined in 48-hour static toxicity tests at 20 ± 2°C.

After 24

and 48 hours, the numbers of immobilised Chironomus riparius were recorded.


5.2.1 48-hour EC50 (immobilisation)

TI-435 : 0.029 mg/L

5.3 Conclusion

5.3.1 Reliability 1





Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...











Remarks



Section A7.4.3.6 Annex Point IIIA XIII.2

Mesocosm study

1 REFERENCE

Officialuse only



1.2.1 Data owner


None



2.1 Guideline study Yes SETAC guidance document on testing procedures for pesticides in freshwater mesocosms (1991) OECD draft guidance document: Freshwater lentic field tests (1996)

2.2 GLP Yes

2.3 Deviations No




3.1.2 Specification

3.1.3 Purity

3.1.4 Appearance



No


3.3.1 Test sediment




Mesocosm study

3.3.2 Test water





Mesocosm study

3.3.4 Test system

3.3.5 Establishment of the test conditions

3.3.6 Application date


0.10, 0.32, 1.0, 3.2, and 10 µg/L TI-435





Mesocosm study

3.3.9 Controls

3.3.10 Duration of the test 14 weeks


3.3.12 Sampling




Mesocosm study


3.3.14 Statistics




Mesocosm study

4 RESULTS



4.3 Effects on aquatic organisms




Mesocosm study

4.4 Effects on freshwater insects

X




Mesocosm study



Toxic effects of TI-435 on a freshwater ecosystem was tested in accordance with the relevant guidelines at the time the study was performed,

No deviations from the guidelines occurred.


5.2.1 EAC (Ecologically Acceptable Concentration)

10 µg/L TI-435

5.3 Conclusion The quality and validity criteria were fulfilled. The used

mesocosm ponds modelled a realistic freshwater ecosystem with sufficient representation from all different trophic levels. The study focussed on the toxicity to aquatic insects, because earlier studies indicated that this might be of concern. Clear concentration-effect relationships were obtained in case of toxic effects. Based on these relationships and univariate statistics NOEC’s as well as an EAC based on recovery data were obtained. The study period was sufficient to demonstrate complete recovery of the affected populations.

5.3.1 Reliability 1 X







Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...







Remarks




Inhibition to microbial activity (terrestrial)

1 REFERENCE

Official

use only

1.1 Reference Keirs, D.C. and Caley, C.Y. (1999): The effects of TI-435 50% WDG

on soil microflora.

Inveresk Research, Tranent EH33 2NE, Scotland; unpublished report

no. 17938




letter of access

None


protection




OECD no. 216 and 217 (drafts of January 1999)

2.2 GLP Yes

2.3 Deviations No


3.1 Test material TI-435 formulated as 50% WDG

3.1.1 Lot/Batch number IW016-046

3.1.2 Specification Not applicable, WDG formulation of TI-435

3.1.3 Purity Not applicable, WDG formulation of TI-435


Product

49.3% TI-435 (active substance), 50.7% inerts


properties

None

3.2 Reference

substance

Yes

Dinoseb acetate, purity: 98.1%


3.3.1 Soil sample A sandy loam soil (64% sand, 26% silt, 10% clay, pH 6.8, 1.1% Corg)

was used as test soil. It was collected from a grassland field in June

1999, sieved (2 mm) and stored at 4°C until the start of the study (July

1999). Before use, the moisture content and the maximum water holding

capacity (MWHC = 47.6 g water/100 dw soil) was determined and the

soil was adjusted to ca. 40% MWHC. The carbon content of the

metabolically active microbial biomass was determined at the start of

the experiment according to ANDERSON AND DOMSCH (1978)1.

3.3.2 Test system





3.3.3 Application of TS The soil was treated at a rate equivalent to 150 g a.s./ha and 5 times this

rate, i.e. 750 g a.s./ha. Assuming a soil depth of 5 cm and a soil density

of 1.5 g/cm3, these rates corresponded to 0.2 mg and 1.0 mg a.s./kg soil.

For further details on application of TS see Table A7_5_1_1-2

3.3.4 Test conditions See Table A7_5_1_1-3

3.3.5 Test parameters and

analytical methods

Soil respiration: After addition of glucose (equivalent to 3.0 g/kg dry

soil), the quantities of carbon dioxide released were measured for at

least 12 hours by means of an infra-red gas analyser.

Nitrogen transformation: After treatment, soil samples in the nitrogen

transformation test were amended with 0.5% (w/w) lucerne meal

(3.5% N, C:N ratio of 11.5:1) to stimulate nitrogen transformation. At

the sampling days, samples of lucerne-amended soil were extracted with

KCl (2 M; 200 mL). Samples of unamended and untreated soil were

either extracted directly or after addition of nitrate to assess nitrification

of unamended soil and the efficiency of nitrate extraction. Extracts and

residues were separated by centrifugation. After filtration, the extracts

were analysed for the total oxidisable nitrogen and nitrite in solution on

an Aquatec 5400 Flow Injection Analyser and Autosampler. Nitrite was

determined by reaction with acidified sulphanilamide to form a diazo

compound and reacted with N-(1-naphthyl)-ethylene-diamine

dihydrochloride to form a purple azo dye compound. Nitrate was

reduced to nitrite (using a cadmium reductor coil) and analysed as

described above. Final nitrate levels were calculated by subtraction of

free nitrite levels formed by reduction of nitrate.


3.3.7 Sampling At intervals of 0-3 h, 7, 14 and 28 days post-application, sub-samples

(ca. 50 g dry weight) of the incubated soils were taken for analysis.

3.3.8 Monitoring of TS

concentration

No

3.3.9 Controls Each test (soil respiration and transformation) included a positive

control (3 soil samples treated with Dinoseb acetate) and a solvent

control (3 soil samples treated with distilled-grade water only).

A further soil sample (nitrate control) was neither treated nor amended

with lucerne meal to determine the nitrogen transformation in

unamended soil and the efficiency of extraction of nitrate.

3.3.10 Statistics Results at each sample point were analysed separately using analysis of

variance (ANOVA). Pairwise comparison between each treated group

and the corresponding control group were compared using Dunnett’s

test (Dunnett, 1955 and 19642). The significance level was p<0.05.

4 RESULTS

4.1 Range finding test Not performed

4.2 Microbial biomass At the start of the experiment the microbial biomass was determined to

be 466.2 mg C/kg dw soil

4.3 Results test

substance





4.3.1 Soil respiration Mean CO2 evolution decreased between day 0 and day 7 and between

day 14 and day 28 in all samples. Mean respiration rates were

comparable between the solvent controls and the two treatments on

day 0 and day 7 and differed only by about 3% at the low dose and 1.5%

at the high dose. On day 14, a significant effect was determined at the

low dose (6% deviation to the control). However, no significant effects

were seen at the higher dose on this sampling date. Moreover, no effects

were seen in both low and high dose at the end of the study after

28 days.

Mean respiration rate

[mL CO2/kg dry soil equiv. and hour]

Sampling Control 150 g a.s./ha 750 g a.s./ha

0-3 hours 10.91 11.27 11.11

7 days 7.79 8.00 7.91

14 days 7.78 8.27* 8.09

28 days 6.24 6.70 6.68

* significantly different from control (p < 0.05)

In summary, short-term respiration of soil treated with TI-435 50%

WDG at up to 750 g a.s./ha deviated from the solvent controls by less

than 25% at each sampling.

4.3.2 Nitrogen

transformation

Mean nitrate levels were comparable at the beginning (about 6.7 mg

N/kg dry soil) and increased over time in all samples up to

approximately 55 mg N/kg dry soil on day 28. Deviations from the

solvent controls were about 3 - 4% at the low dose and between 4 and

14% at the high dose. No significant effects were determined.

Concentration of nitrate

[mg N/kg dry soil equiv.]

Sampling Control 150 g a.s./ha 750 g a.s./ha

0-3 hours 6.6 6.8 7.5

7 days 22.6 23.5 23.5

14 days 35.1 36.5 37.6

28 days 53.5 55.0 55.4

In summary, nitrogen transformation in soil treated with TI-435 50%

WDG at up to 750 g a.s./ha deviated from the solvent controls by less

than 25% at each sampling.


4.4.1 Test with reference

substance (positive

control)

Soil respiration: In the soils treated with Dinoseb acetate, the rates of

short-term respiration were between 42.8% and 65.7% lower than the

rates found in the solvent controls, during the 28-day experiment. All

values were statistically significant (p<0.05).

Nitrogen transformation: In the soils treated with Dinoseb acetate, the

nitrate concentrations were significant lower (p<0.05) compared to the

controls (42.5% after 14 days and 32.5% after 28 days).





4.4.2 Nitrate control Levels of nitrate in soil which had not been amended with lucerne or

treated with the test substance, increased from 5.2 mg to 23.3 mg N/kg

over the incubation period, demonstrating significant nitrification

activity.


5.1 Materials and

methods

The effects of TI-435 50% WDG on respiration and nitrogen

transformation of soil microflora under aerobic conditions was

determined according to OECD 216 and 217. TI-435 50% WDG was

incorporated into a sandy loam soil at treatment rates of 0.2 mg a.s./kg

dw soil (equivalent to the maximum field application rate, i.e. 150 g

a.s./ha) and 1.0 mg a.s./kg dw soil

.

5.2 Results and

discussion

5.3 Conclusion Validity criteria can be considered as fulfilled

Based on these results, TI-435 is no expected to adversely affect the soil

microflora when applied up to 750 g a.s./ha (equivalent to 1.0 mg a.s./kg

dry soil).

5.3.1 Reliability 1






Date 2007-01-05

Materials and Methods applicant’s version is acceptable although testing was performed with a product

other than the biocidal product SPU-01850-I. However, despite the differing

composition of both products, the results obtained here are considered acceptable

to assess the potential adverse effects of the active substance chlothianidin on soil

micro-organisms.

The soil was treated at a rate equivalent to 150 g a.s./ha and 5 times this rate, i.e.

750 g a.s./ha. Assuming a soil depth of 10 cm and a soil density of 1500 kg/m3

dry weight, (as decided on TM III/06) these rates corresponded to 0.1 mg and

0.5 mg a.s./kg dry soil.

Results and discussion applicant's version is adopted.


Reliability 1


Remarks none

COMMENTS FROM ...













Remarks



Table A7_5_1_1-1: Test system

Criteria Details

Culturing apparatus 2.5 L plastic containers

Number of vessels / concentration 3

Amount of soil/culturing apparatus 1.2 kg

Aeration device No

Measuring equipment Infra-red gas analyser (respirometer) to determine the

soil respiration activity

Photometer to determine nitrate and nitrite extracted

from soil

Test performed in closed vessels No, plastic containers were sealed with a snap-top lid

containing holes to maintain aeration

Table A7_5_1_1-2: Application of test substance

Criteria Details

Application procedure Applied in aqueous solution

Carrier Distilled-grade water

Concentration of a.s. in treatment solutions 100 mg a.s./L (5 x maximum field application rate)

20 mg a.s./L (maximum field application rate)

Liquid carrier control Yes

Distilled-grade water

Procedure Soil replicates were treated by evenly dispensing

12.0 mL aliquots of the treatment solutions onto the

soil surface in the test containers. The soil was mixed

thoroughly during and after treatment. Control

samples were treated in the same manner but with

12.0 mL of distilled-grade water only.

Table A7_5_1_1-3: Test conditions

Criteria Details

Organic substrate After treatment, soil samples for the nitrogen

transformation test were amended with ca. 0.5%

(w/w) lucerne meal and thoroughly mixed.

Incubation temperature 20 ± 2°C

Soil moisture Ca. 40% maximum water holding capacity (MWHC),

i.e. 19.0 g water/100 dw soil; The soil moisture

content was controlled at weekly intervals and re-

adjusted to 40% MWHC using distilled-grade water.

Method of soil incubation As bulk; 3 soil samples per concentration/solvent

control/positive control and per test (respiration and

nitrogen transformation), 1 untreated and unamended

soil sample (nitrate control)

Aeration No


Section A7.5.1.2/01 Annex Point IIIA XIII.3.2

Acute toxicity to earthworms or other soil non-target macro-organisms

1 REFERENCE

Officialuse only

1.1 Reference (1998b):

1.2 Data protection Yes 1.2.1 Data owner 1.2.2 Companies with

letter of access



2.1 Guideline study Yes OECD No. 207 (1984) and Directive 87/302/EEC, Part C

2.2 GLP Yes

2.3 Deviations

3 METHOD

3.1 Test material


3.1.2 Specification

3.1.3 Purity







3.3.1 Preparation of the test substance

3.3.2 Application of the test substance


3.3.4 Test system






3.3.6 Test duration 14 days

3.3.7 Test parameter Mortality and body weight

3.3.8 Examination

3.3.9 Monitoring of test substance concentration

3.3.10 Statistics

4 RESULTS

4.1 Filter paper test

4.2 Rangefinder test

4.2.1 Concentration

4.2.2 Proposed LC50

4.3 Definite test


0, 10, 18, 32, 56 and 100 mg/kg dw artificial soil


4.3.3 Concentration / effect curve

4.3.4 Other effects


4.4.1 Mortality

4.4.2 Number/ percentage of earthworms showing adverse effects








4.5.2 Results



The acute toxicity of TI-435 technical to earthworms was tested according to OECD No. 207 (1984) and Directive 87/302/EEC, Part C. Eisenia foetida were exposed to nominal concentrations of 0, 10, 18, 32, 56 and 100 mg a.s./kg dw artificial soil and effects on mortality and body weight were observed.


After 7 days, there was 100% mortality in 56 and 100 mg/kg treatments, with some mortality in all TI-435 treatments. After 14 days, there was 100% mortality in 32, 56 and 100 mg/kg treatments, with some mortality in all lower TI-435 treatment replicates. There were no control mortalities. Surviving earthworms in the 10 and 18 mg/kg treatments showed a significant reduction in mean body weight at the end of the test.

5.2.1 NOEC < 10 mg/kg dw soil (based on reduction in body weight)

5.2.2 LC50 (7 days exposure)

18.58 mg/kg dw soil (calculated)


13.21 mg/kg dw soil (calculated)


32 mg/kg dw soil (nominal)


.

5.3.1 Reliability 1





Date



Conclusion

Reliability

Acceptability

Remarks












Remarks



Section A7.5.1.2/02 and A7.5.2.1/02 Annex Point IIIA XIII.3.2

Acute toxicity to earthworms or other soil non-target macro-organisms Reproduction study with earthworms or other soil non-target macro-organisms

1 REFERENCE

Officialuse only



letter of access




2.2 GLP Yes

2.3 Deviations No

3 METHOD



3.1.2 Specification

3.1.3 Purity














3.3.4 Test system



3.3.7 Test parameter Mortality and reproduction

3.3.8 Examination

3.3.10 Statistics

4 RESULTS


4.2 Soil test


0.01, 0.032, 0.1, 0.32 and 1.0 mg/kg dw artificial soil



4.2.4 Other effects


4.3.1 Mortality

4.3.2 Reproduction



4.4.2 Results







The toxicity of TI-435 to collembola was tested according to ISO/FDIS 11267 (1998). Folsomia candida were exposed to nominal concentrations of 0.01, 0.032, 0.1, 0.32 and 1.0 mg/kg dw artificial soil and effects on mortality and reproduction were observed.


5.2.1 LC50 (mortality) 1.02 mg/kg dw soil (calculated)

5.2.2 LOEC (mortality) 1.0 mg/kg dw soil (nominal)

5.2.3 NOEC (mortality) 0.32 mg/kg dw soil (nominal)

5.2.4 EC50 (reproduction) 0.76 mg/kg dw soil (calculated) X


1.0 mg/kg dw soil (nominal)




5.3.1 Other Conclusions

5.3.2 Reliability 1







Date



Conclusion

Reliability

Acceptability

Remarks








Remarks




Section 7.5.1.3/01 and

7.5.2.2/01 Annex Point IIIA XIII.3.4

Acute toxicity to plants

Long-term test with terrestrial plants

1 REFERENCE

Official

use only

1.1 Reference Brignole, A.J.; Porch, J.R. and Krueger, H.O. (2000a): TI-435 50%

WDG: A toxicity test to determine the effects of the test substance on

seedling emergence of ten species of plants.

Wildlife International, Ltd. Easton, Maryland 21601, USA; unpublished

report of project 197-126




letter of access

None


protection




US EPA OPPTS 850.4100 and 850.4225

2.2 GLP Yes

2.3 Deviations No

3 METHOD

3.1 Test material TI-435 50% WDG





Product



properties

None

3.1.6 Method of analysis The spray mixture of TI-435 50% WDG was diluted in

acetonitrile:NANOpure® water (10:90), as necessary, and analysed by

direct injection on the HPLC with UV detection.

LOQ: 1.0 mg a.s./L


solution for poorly

soluble or volatile

test substances

Not relevant. The active substance TI-435 is neither poorly soluble nor

volatile.

3.3 Reference

substance

No

3.4 Blank formulation Yes

Tan powder (TI-435 50WDG Blank Formulation)



spray mixtures

The spray mixture for the test concentration (240 ppm a.s.) was

prepared by diluting 0.4875 g of the test substance to a volume of







1000 mL with reverse osmosis water. Accordingly, the spray mixture

for the formulation blank was prepared by diluting 0.4878 g of the

formulation blank to a volume of 1000 mL with reverse osmosis water.

3.5.2 Test plants

3.5.3 Test system

3.5.4 Test conditions The soil surfaces were sprayed with TI-435 50% WDG at the nominal

concentration of equivalent 225 g a.s./ha (spray volume equivalent to

940 L/ha), a formulation blank and a water control. For further details

see Table A7_5_1_3_01-3.


3.5.6 Test parameter Emergence, growth and changes in general condition of seedlings

Observations on day 10 were made to document seedlings emergence.

Observations on day 14 were made to document seedling emergence

and growth, and to determine changes in general condition of seedlings.

The growth of emerged seedling was evaluated by assessing the height

and dry weight. Seedling condition was described by noting possible

signs of phytotoxicity (i.e. necrosis, leaf wrinkle, chlorosis, plant

lodging or plant stunting).

3.5.7 Sampling Analytical samples were collected immediately after spray mixtures

were prepared. Triplicate samples were collected from the 225 g a.s./ha

treatment group spray mixture in order to verify the test concentration

and homogeneity in the dosing solution. Single samples were collected

from the formulation blank and the negative control spray mixtures.


of the plant material

Not relevant. Only height and dry weight of living seedlings were

determined at test termination.

3.5.9 Statistics A t-test was used to determine if the mean emergence, height or weight

of the treatment group differed significantly from the mean of the

pooled controls (water control and formulation blank). Dunnett’s test

was used in establishing the NOEC by determining significant

differences between the treatments and the water control.

4 RESULTS

4.1 Results test

substance

4.1.1 Applied initial

concentration

Analysis of the spray mixture showed that the test concentration was in

the range of 218 - 234 ppm a.s., i.e. 90.9 – 97.4% of nominal (mean:

94.9%).

4.1.2 Phytotoxicity rating There were no apparent adverse treatment-related effects on any of the

10 plant species tested.

4.1.3 Plant height The heights of treated plants at test termination (day 14) were in the

range of 96 - 106% of the pooled controls.

4.1.4 Plant dry weights The dry weights of treated plants at test termination (day 14) were in the


4.1.5 Emergence The emergence of treated plants at test termination (day 14) was in the


4.1.6 Number of dead None for all species







plants

4.1.7 Effect data For effect data see Table A7_5_1_3_01-4.

Differences in seedling emergence and growth between the 225 g a.s./ha

treatment group means and the pooled control means were minor and

not statistically significant (p>0.05). Therefore, the application of

TI-435 50% WDG at a rate equivalent to 225 g a.s./ha was

determined to be the NOEC for emergence and growth of all species

tested.

4.1.8 Concentration /

response curve

Not relevant (see 4.1.7)

4.1.9 Other effects None for all species and treatment groups



concentration

Analysis of the formulation blank and of the water control showed that

neither contained TI-435.

4.2.2 Number/

percentage of plants

showing adverse

effects

Neither seedlings of the formulation blank nor seedlings of the water

control showed adverse effects.

4.3 Test with

reference

substance

Not performed


5.1 Materials and

methods

Toxic effects of TI-435 50% WDG on seedling emergence and growth

of plants were tested according to US EPA OPPTS

850.4100 and 850.4225. No deviations from the guidelines occurred.

5.2 Results and

discussion

X

5.2.1 NOEC 225 g a.s./ha for emergence and growth of all species tested

5.3 Conclusion There were no observed treatment-related effects on any of the test

species.

X

5.3.1 Reliability 1












Date 2005-08-16

Materials and Methods Applicant’s version is acceptable.

Results and discussion There were no apparent adverse treatment-related effects on 9 of the 10 plant

species tested. For Lycopersicon esculentum however a reduction in emergence of

22 % compared to the control was observed at the only tested concentration of

225 g a.s/ha.. However, the difference to the control was not statistically

significant. Therefore, the overall NOEC from this study is 225 g a.s./ha.

Conclusion For all tested species a NOEC of 225 g a.s./ha can be derived from this study for

emergence and growth, as no statistically significant effects compared to the

control were found.

Reliability 1


Remarks The performed test is an acute plant test and can not be submitted as acute and

long-term study.










Remarks



Family Species Common name Source (seed)

Dicotyledonae Leguminosae Phaseolus

vulgaris

Pinto bean Meyer Seed Co.,

Baltimore, MD

Compositae Lactuca sativa Lettuce Territorial Seed Co.,

Cottage Grove, OR

Cruciferae Brassica olearaea Cabbage Meyer Seed Co.,

Baltimore, MD

Leguminosae Glycine max Soybean Johnny’s Selected

Seeds, Albion, ME

Solanaceae Lycopersicon

esculentum

Tomato Meyer Seed Co.,

Baltimore, MD

Cruciferae Raphanus sativus Radish Meyer Seed Co.,

Baltimore, MD

Monocotyledonae Liliaceae Allium cepa Onion Territorial Seed Co.,

Cottage Grove, OR

Gramineae Lolium perenne Ryegrass Meyer Seed Co.,

Baltimore, MD

Gramineae Zea mays Field corn Johnny’s Selected

Seeds, Albion, ME

Gramineae Triticum aestivum Wheat Arrowhead Mills

Inc., Hereford, TX


Criteria Details

Test type Greenhouse

Container type Plastic pots (16 cm diameter, 11 cm-deep)

Seed germination potential Not stated

Identification of the plant species Seeds were impartially assigned to pre-labelled

growth pots on the day of test initiation.

Number of replicates 4

Numbers of plants per replicate per dose 10

Date of planting October 12, 1999

Plant density 10 seeds per pot put into 10 single holes in the soil

surface (surface: 201 cm²)

Date of test substance application October 12, 1999

High of plants at application Application was performed directly after planting the

seeds.

Date of phytotoxicity rating or harvest See 3.5.6




Criteria Details

Test type Seedling emergence and growth

Method of application A pressurised spray applicator was used to spray the soil

surface in pots.

Number of applications: 1

Application levels 225 g a.s./ha (spray volume equivalent to 940 L/ha)

Dose rate 225 g a.s./ha

Substrate characteristics sand soil composed of industrial quartz sand, kaolinite clay

and peat (ratio 50:4:5)

soil analysis showed 91% sand, 2% silt, 7% clay, 2.4%

organic matter and a pH of 7.8

limestone (to buffer the pH) and a slow-release fertiliser

were added

Watering of the plants Water lost through transpiration and evaporation replaced by

subirrigation with well water

Temperature 23 to 28°C

Photoperiod 12 hours

Light regime 411 to 7518 kW/m2 light intensity

(natural sunlight not supplemented with artificial lighting)

Relative humidity 21 to 89%

Wind volatility Not relevant, test performed in greenhouse.

Observation periods and duration of test Observations were made on day 10 (seedlings emergence)

and day 14 (seedling emergence, growth and changes in

general condition of seedlings).

Test duration: 15 days

Pest control No

Any other treatments and procedures No

Table A7_5_1_3_01-4: Emergence and effective phytotoxicity of treated seeds (225 g a.s./ha) on day 14

Plant Absolute numbers Per cent relative to pooled controls

Plant

height [cm]

Plant dry

weight [mg]

Emergence Plant

height [%]

Plant dry

weights [%]

Emergence

[%]

Pinto bean 20.2 245 9.75 102 110 99

Cabbage 5.1 9.2 9.5 106 98 107

Field corn 40.0 176 9.75 97 101 103

Lettuce 4.3 3.6 9.25 103 113 97

Onion 7.2 2.9 8.75 105 98 101

Radish 6.0 22 8.25 97 111 94

Ryegrass 8.0 2.1 10.0 99 95 110

Soybean 26.1 313 10.0 97 94 100

Tomato 4.2 6.9 5.75 106 100 78

Wheat 25.5 26 9.75 96 104 100

Table A7_5_1_3_01-5: Validity criteria for terrestrial plant toxicity according to EPA OPPTS 850.4100

Fulfilled Not fulfilled

Adverse effect > 25% on one or more plant species (EPA) x



Section 7.5.1.3/02 Annex Point IIIA XIII.3.4


1 REFERENCE

Official

use only

1.1 Reference Brignole, A.J.; Porch, J.R. Krueger, H.O. and Kendall, T.Z. (2000b): TI-

435 50% WDG: A toxicity test to determine the effects of the test

substance on vegetative vigor of ten species of plants.






letter of access

None


protection




US EPA OPPTS 850.4150

2.2 GLP Yes

2.3 Deviations No

3 METHOD






Product



properties

None


acetonitrile:NANOpure® water (10:90), as necessary, and analysed

by direct injection on the HPLC with UV detection.

LOQ: 1.0 mg a.s./L


solution for poorly

soluble or volatile

test substances


volatile.

3.3 Reference

substance

No





spray mixtures










3.5.2 Test plants See Table A7_5_1_3_02-1


3.5.4 Test conditions A spray application of TI-435 50% WDG at a nominal concentration of

equivalent to 225 g a.s./ha (spray volume equivalent to 940 L/ha), a

formulation blank and a water control was made to the plant foliage. For

further details see Table A7_5_1_3_02-3.


3.5.6 Test parameter Plant growth and condition (phytotoxicity)

Plant growth was evaluated by assessing the height and dry weight.

Plant condition was described by noting possible signs of phytotoxicity

(i.e. necrosis, leaf wrinkle, chlorosis, plant lodging or plant stunting).

Plant heights and condition were observed on day 0 (prior to

application), days 7 and 14. Shoot dry weights were determined at test

termination.








Not relevant. Only height and dry weight of plants were determined.

3.5.9 Statistics

4 RESULTS

4.1 Results test

substance


concentration



96.8%).



4.1.3 Plant height The heights of treated plants at day 7 and at day 14 were in the range of

96 - 105% and 97 - 105% of the pooled controls, respectively.

4.1.4 Plant dry weights The dry weights of treated plants at day 14 were in the range of

91 - 109% of the pooled controls.

4.1.5 Number of dead

plants

None for all species


Differences between treatment group means and the pooled control





means were minor and not statistically significant (p>0.05).

Therefore, the application of TI-435 50% WDG at 225 g a.s./ha was

determined to be the NOEC for vegetative vigor in all species tested.


response curve

Not relevant (see 4.1.6)




concentration



4.2.2 Number/


showing adverse

effects

Neither plants of the formulation blank nor plants of the water control

showed adverse effects.

4.3 Test with

reference

substance

Not performed


5.1 Materials and

methods

Toxic effects of TI-435 50% WDG on vegetative vigor of

plants were tested according to US EPA OPPTS 850.4150. No

deviations from the guideline occurred.

5.2 Results and

discussion

5.2.1 NOEC 225 g a.s./ha for vegetative vigor of all species tested


species.

5.3.1 Reliability 1






Date 04.02.2005

Materials and Methods Applicant’s version is acceptable.

Results and discussion Applicant's version can be adopted.

Conclusion Applicant's version can be adopted.

Reliability 1






Remarks










Remarks



Table A7_5_1_3_02-1: Test plants



vulgaris


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


esculentum


Baltimore, MD


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


Inc., Hereford, TX


Criteria Details




Identification of the plant species Seedlings were randomly assigned to the treatment

and control groups and labelled with the species

name, project number, treatment group, replicate

designation and plant number.


Numbers of plants per replicate per dose 5 plants, each in a separate pot

Date of planting September 27 to October 7, 1999

Plant density 1 plant per pot


High of plants at application Pinto bean: 14.4 cm, cabbage: 8.3 cm, field corn:

18.6 cm, lettuce: 4.98 cm, onion: 8.4 cm, radish:

8.8 cm, ryegrass: 11.5 cm, soybean: 13.4 cm, tomato:

5.4 cm, wheat: 18.9 cm (mean values)





Criteria Details

Test type Vegetative vigor

Method of application pressurised spray applicator

spray of plant foliage









were added









Observation periods and duration of test Plant heights and condition were observed on day 0 (prior to

application), days 7 and 14. Shoot dry weights were



Pest control No


Table A7_5_1_3_02-4: Plant heights and dry weights of treated plants (225 g a.s./ha)


Plant height

at day 7

[cm]

Plant height

at day 14

[cm]

Plant dry

weight at

day 14 [mg]

Plant height

at day 7

[%]

Plant height at

day 14

[%]

Plant dry

weight at

day 14 [%]

Pinto bean 19.2 26.1 1.43 96 98 101

Cabbage 12.7 16.8 0.85 105 104 101

Field corn 44.4 66.9 1.02 100 100 96

Lettuce 9.0 13.1 0.16 103 105 91

Onion 13.8 16.0 0.022 96 100 100

Radish 11.6 14.6 0.179 99 101 104

Ryegrass 15.9 21.4 0.068 100 100 96

Soybean 22.7 31.1 1.34 98 97 98

Tomato 10.2 18.1 0.709 100 98 95

Wheat 31.2 34.9 0.226 101 101 109

Table A7_5_1_3_02-5: Validity criteria for terrestrial plant toxicity according to

EPA OPPTS 850.4150 (vegetative vigor test)





Reproduction study with earthworms or other soil non-target macro-organisms

1 REFERENCE

Officialuse only



letter of access None




2.2 GLP Yes

2.3 Deviations None

3 METHOD



3.1.2 Specification

3.1.3 Purity














3.3.4 Test system


3.3.6 Test duration 28 days (adult earthworms) + 28 days (development of offspring)

3.3.7 Test parameter Mortality, body weight and reproduction

3.3.8 Examination


3.3.10 Statistics

4 RESULTS

4.1 Results of test substance

4.1.1 Application rate

4.1.2 Mortality

4.1.3 Body weights

4.1.4 Reproduction

4.1.5 Other effects


4.2.1 Mortality

4.2.2 Body weights





4.2.3 Reproduction


4.3.1 Concentration

4.3.2 Mortality

4.3.3 Body weights

4.3.4 Reproduction



The reproduction toxicity of TI-435 50% WDG to earthworms was tested

Earthworms were exposed to 150 g a.s./ha and five times this rate (750 g a.s./ha) and effects on

mortality, body weight and reproduction were observed. No major deviations from the guidelines occurred.


5.3 Conclusion At application rates up to the maximum field application rate, no negative effects on earthworm reproduction are to be expected. Validity criteria can be considered as fulfilled

5.3.1 Reliability 1







Date



Conclusion

Reliability

Acceptability

Remarks








Remarks







1 REFERENCE

Official use only



1.2.1 Data owner





2.2 GLP Yes

2.3 Deviations No

3 METHOD



3.1.2 Specification

3.1.3 Purity















3.3.4 Test system



3.3.7 Test parameter Mortality and reproduction

3.3.8 Examination


3.3.10 Statistics

4 RESULTS


4.2 Soil test


0.01, 0.032, 0.1, 0.32 and 1.0 mg/kg dw artificial soil



4.2.4 Other effects


4.3.1 Mortality

4.3.2 Reproduction








4.4.2 Results



The toxicity of TI-435 to collembola was tested . Folsomia candida were exposed to nominal

concentrations of 0.01, 0.032, 0.1, 0.32 and 1.0 mg/kg dw artificial soil and effects on mortality and reproduction were observed.


5.2.1 LC50 (mortality) 1.02 mg/kg dw soil (calculated)

5.2.2 LOEC (mortality) 1.0 mg/kg dw soil (nominal)

5.2.3 NOEC (mortality) 0.32 mg/kg dw soil (nominal)

5.2.4 LC50 (reproduction) 0.76 mg/kg dw soil (calculated)






5.3.1 Other Conclusions

5.3.2 Reliability 1





Use separate "evaluation boxes" to provide transparency as to the comments and views submitted


Date



Conclusion

Reliability

Acceptability

Remarks








Remarks

Sumitomo Chemical Takeda Agro Co. Ltd. TI-435 May 2008



1 REFERENCE

Officialuse only



letter of access None



2.1 Guideline study •

2.2 GLP Yes

2.3 Deviations None

3 METHOD



3.1.2 Specification

3.1.3 Purity





3.2.1


3.3.1 Trial location and layout





identification. Deeper living earthworms were extracted by evenly





4 RESULTS

4.1 Results of test substance





4.2 Results of toxic reference substance







A supervised field trial was conducted over twelve months to determine the effects of TI-435 50% WDG on earthworms. TI-435 50% WDG was applied at a nominal rate of 75 g a.s./ha, 112 g a.s./ha and 225 g a.s./ha to plots two, three and four, respectively.


Application of TI-435 50% WDG had no significant detrimental effect (to at least the 5% level) on the number of earthworms, or their biomass, following application at rates from 75 to 225 g a.s./ha, compared to the untreated control.

5.3 Conclusion Considering the results of the statistical analysis and the variability of earthworm abundance in natural soils, this study indicates that earthworm populations were not affected (to at least the 5% level) by the application of TI-435 50% WDG at rates from 75 to 225 g a.s./ha, compared to an untreated control, for a period up to one year after application.

5.3.1 Reliability 1









Date



Conclusion

Reliability

Acceptability

Remarks








Remarks






1 REFERENCE

Official

use only

1.1 Reference Brignole, A.J.; Porch, J.R. and Krueger, H.O. (2000a): TI-435 50%

WDG: A toxicity test to determine the effects of the test substance on

seedling emergence of ten species of plants.






letter of access

None


protection




US EPA OPPTS 850.4100 and 850.4225

2.2 GLP Yes

2.3 Deviations No

3 METHOD






Product



properties

None


acetonitrile:NANOpure® water (10:90), as necessary, and analysed

by direct injection on the HPLC with UV detection.

LOQ: 1.0 mg a.s./L


solution for poorly

soluble or volatile

test substances


volatile.

3.3 Reference

substance

No





spray mixtures










3.5.2 Test plants

3.5.3 Test system

3.5.4 Test conditions The soil surfaces were sprayed with TI-435 50% WDG at the nominal

concentration of equivalent 225 g a.s./ha (spray volume equivalent to

940 L/ha), a formulation blank and a water control. For further details

see Table A7_5_1_3_01-3.


3.5.6 Test parameter Emergence, growth and changes in general condition of seedlings

Observations on day 10 were made to document seedlings emergence.

Observations on day 14 were made to document seedling emergence

and growth, and to determine changes in general condition of seedlings.

The growth of emerged seedling was evaluated by assessing the height

and dry weight. Seedling condition was described by noting possible

signs of phytotoxicity (i.e. necrosis, leaf wrinkle, chlorosis, plant

lodging or plant stunting).








Not relevant. Only height and dry weight of living seedlings were


3.5.9 Statistics A t-test was used to determine if the mean emergence, height or weight

of the treatment group differed significantly from the mean of the

pooled controls (water control and formulation blank). Dunnett’s test

was used in establishing the NOEC by determining significant

differences between the treatments and the water control.

4 RESULTS

4.1 Results test

substance


concentration


the range of 218 - 234 ppm a.s.,


plant species tested.

4.1.3 Plant height The heights of treated plants at test termination (day 14) were in the


4.1.4 Plant dry weights The dry weights of treated plants at test termination (day 14) were in the


4.1.5 Emergence The emergence of treated plants at test termination (day 14) was in the



plants







Differences in seedling emergence and growth between the 225 g a.s./ha

treatment group means and the pooled control means were minor and

not statistically significant (p>0.05). Therefore, the application of

TI-435 50% WDG at a rate equivalent to 225 g a.s./ha was determined

to be the NOEC for emergence and growth of all species tested.


response curve




concentration



4.2.2 Number/


showing adverse

effects

Neither seedlings of the formulation blank nor seedlings of the water

control showed adverse effects.

4.3 Test with

reference

substance

Not performed


5.1 Materials and

methods

Toxic effects of TI-435 50% WDG on seedling emergence and growth

of of plants were tested according to US EPA OPPTS

850.4100 and 850.4225. No deviations from the guidelines occurred.

5.2 Results and

discussion

5.2.1 NOEC 225 g a.s./ha for emergence and growth of all species tested


species.

5.3.1 Reliability 1










Date 2005-05-25

Materials and Methods Applicant's version is not acceptable for long term testing because the submitted

study is an acute plant test

Results and discussion Applicant's version can not be adopted for long term testing because the

submitted study is an acute plant test .

Conclusion Applicant's version can not be adopted for long term testing because the


Reliability -Not applicable due to study concept.

Acceptability Not acceptable; the performed test is an acute plant test and can not be submitted

as long-term study. No data were submitted for Section A7.5.2.2 Long-term test

with terrestrial plants. A long term test with terrestrial plants is not required.

Remarks










Remarks



_5_1_3_01-1: Test plants



vulgaris


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


esculentum


Baltimore, MD


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


Inc., Hereford, TX


Criteria Details




Identification of the plant species Seeds were impartially assigned to pre-labelled

growth pots on the day of test initiation.


Numbers of plants per replicate per dose 10

Date of planting October 12, 1999

Plant density 10 seeds per pot put into 10 single holes in the soil

surface (surface: 201 cm²)


High of plants at application Application was performed directly after planting the

seeds.





Criteria Details

Test type Seedling emergence and growth

Method of application A pressurised spray applicator was used to spray the soil

surface in pots.









were added









Observation periods and duration of test Observations were made on day 10 (seedlings emergence)

and day 14 (seedling emergence, growth and changes in

general condition of seedlings).


Pest control No


Table A7_5_1_3_01-4: Emergence and effective phytotoxicity of treated seeds (225 g a.s./ha) on day 14


Plant

height [cm]

Plant dry

weight [mg]

Emergence Plant

height [%]

Plant dry

weights [%]

Emergence

[%]

Pinto bean 20.2 245 9.75 102 110 99

Cabbage 5.1 9.2 9.5 106 98 107

Field corn 40.0 176 9.75 97 101 103

Lettuce 4.3 3.6 9.25 103 113 97

Onion 7.2 2.9 8.75 105 98 101

Radish 6.0 22 8.25 97 111 94

Ryegrass 8.0 2.1 10.0 99 95 110

Soybean 26.1 313 10.0 97 94 100

Tomato 4.2 6.9 5.75 106 100 78

Wheat 25.5 26 9.75 96 104 100

Table A7_5_1_3_01-5: Validity criteria for terrestrial plant toxicity according to EPA OPPTS 850.4100







1 REFERENCE

Official

use only

1.1 Reference Brignole, A.J.; Porch, J.R. Krueger, H.O. and Kendall, T.Z. (2000b): TI-

435 50% WDG: A toxicity test to determine the effects of the test

substance on vegetative vigor of ten species of plants.






letter of access

None


protection




US EPA OPPTS 850.4150

2.2 GLP Yes

2.3 Deviations No

3 METHOD






Product



properties

None


acetonitrile:NANOpure® water (10:90), as necessary, and analysed by

direct injection on the HPLC with UV detection.

LOQ: 1.0 mg a.s./L


solution for poorly

soluble or volatile

test substances


volatile.

3.3 Reference

substance

No





spray mixtures





formulation blank to a volume of 1000 mL with reverse osmosis





water.

3.5.2 Test plants See Table A7_5_1_3_02-1


3.5.4 Test conditions A spray application of TI-435 50% WDG at a nominal concentration of

equivalent to 225 g a.s./ha (spray volume equivalent to 940 L/ha), a

formulation blank and a water control was made to the plant foliage.

For further details see Table A7_5_1_3_02-3.


3.5.6 Test parameter Plant growth and condition (phytotoxicity)

Plant growth was evaluated by assessing the height and dry weight.

Plant condition was described by noting possible signs of

phytotoxicity (i.e. necrosis, leaf wrinkle, chlorosis, plant lodging or

plant stunting). Plant heights and condition were observed on day 0

(prior to application), days 7 and 14. Shoot dry weights were



were prepared. Triplicate samples were collected from the 225 g

a.s./ha treatment group spray mixture in order to verify the test

concentration and homogeneity in the dosing solution. Single samples

were collected from the formulation blank and the negative control

spray mixtures.



Not relevant. Only height and dry weight of plants were determined.

3.5.9 Statistics For each species, an assessment was made as to whether the test

concentration was a NOEC. Plant growth was assessed by comparing

treatment group means for shoot height and dry weight. Evaluation of

plant condition was also used in determining treatment related effects.

Although group mean plant scores were calculated, statistical

comparison of the means was not performed since condition is a

qualitative measurement (relative not quantitative differences).

A t-test was used to determine if the mean height or dry weight of the

treatment group differed significantly from the mean of the pooled

controls (water control and formulation blank).

4 RESULTS

4.1 Results test

substance


concentration



96.8%).



4.1.3 Plant height The heights of treated plants at day 7 and at day 14 were in the range of

96 - 105% and 97 - 105% of the pooled controls, respectively.

4.1.4 Plant dry weights The dry weights of treated plants at day 14 were in the range of 91 -

109% of the pooled controls.


plants








means were minor and not statistically significant (p>0.05). Therefore,

the application of TI-435 50% WDG at 225 g a.s./ha was determined to

be the NOEC for vegetative vigor in all species tested.


response curve




concentration



4.2.2 Number/


showing adverse

effects

Neither plants of the formulation blank nor plants of the water control

showed adverse effects.

4.3 Test with

reference

substance

Not performed


5.1 Materials and

methods

Toxic effects of TI-435 50% WDG on vegetative vigor of

plants were tested according to US EPA OPPTS 850.4150. No

deviations from the guideline occurred.

5.2 Results and

discussion

There were no apparent adverse treatment-related effects on any of the

10 plant species tested after application of equivalent 225 g a.s./ha.


means were minor and not statistically significant (p>0.05).

5.2.1 NOEC 225 g a.s./ha for vegetative vigor of all species tested


species.

5.3.1 Reliability 1










Date 2005-05-25

Materials and Methods Applicant's version is not acceptable for long term testing because the submitted

study is an acute plant test.

Results and discussion Applicant's version can not be adopted for long term testing because the

submitted study is an acute plant test

Conclusion Applicant's version can not be adopted for long term testing because the


Reliability Not applicable due to study concept.

Acceptability Non acceptable;the performed test is an acute plant test and can not be submitted

as acute and longterm study. No data were submitted for Section A7.5.2.2/02

Long-term test with terrestrial plants. A long term test with terrestrial plants is

not required.

Remarks










Remarks



Table A7_5_1_3_02-1: Test plants



vulgaris


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


esculentum


Baltimore, MD


Baltimore, MD


Cottage Grove, OR


Baltimore, MD


Seeds, Albion, ME


Inc., Hereford, TX


Criteria Details




Identification of the plant species Seedlings were randomly assigned to the treatment

and control groups and labelled with the species

name, project number, treatment group, replicate

designation and plant number.


Numbers of plants per replicate per dose 5 plants, each in a separate pot

Date of planting September 27 to October 7, 1999

Plant density 1 plant per pot


High of plants at application Pinto bean: 14.4 cm, cabbage: 8.3 cm, field corn:

18.6 cm, lettuce: 4.98 cm, onion: 8.4 cm, radish:

8.8 cm, ryegrass: 11.5 cm, soybean: 13.4 cm,

tomato: 5.4 cm, wheat: 18.9 cm (mean values)





Criteria Details

Test type Vegetative vigor

Method of application pressurised spray applicator

spray of plant foliage









were added









Observation periods and duration of test Plant heights and condition were observed on day 0 (prior to

application), days 7 and 14. Shoot dry weights were



Pest control No


Table A7_5_1_3_02-4: Plant heights and dry weights of treated plants (225 g a.s./ha)


Plant height

at day 7

[cm]

Plant height

at day 14

[cm]

Plant dry

weight at

day 14 [mg]

Plant height

at day 7

[%]

Plant height

at day 14

[%]

Plant dry

weight at

day 14 [%]

Pinto bean 19.2 26.1 1.43 96 98 101

Cabbage 12.7 16.8 0.85 105 104 101

Field corn 44.4 66.9 1.02 100 100 96

Lettuce 9.0 13.1 0.16 103 105 91

Onion 13.8 16.0 0.022 96 100 100

Radish 11.6 14.6 0.179 99 101 104

Ryegrass 15.9 21.4 0.068 100 100 96

Soybean 22.7 31.1 1.34 98 97 98

Tomato 10.2 18.1 0.709 100 98 95

Wheat 31.2 34.9 0.226 101 101 109

Table A7_5_1_3_02-5: Validity criteria for terrestrial plant toxicity according to

EPA OPPTS 850.4150 (vegetative vigor test)



Sumitomo Chemical clothianidin (TI-435) August 2008


Section A7.5.4.1 (01) Annex Point IIIA XIII.3.1

Acute toxicity to honeybees and other beneficial arthropods, e.g. predators

1 REFERENCE

Official use only



1.2.1 Data owner




2.1 Guideline study

2.2 GLP Yes

2.3 Deviations


3.1 Test material


3.1.2 Specification

3.1.3 Purity









3.4.3 Test system







3.4.6 Test parameter Behaviour Mortality Metamorphosis

Body weight of adult beetles

4 RESULTS

4.1 Limit Test

4.2 Test substance

4.3 Controls








The effect of TI-435 on larvae of Poecilus cupreus was tested under extended laboratory conditions. The larvae were exposed to the test substance mixed into soil at concentrations of 0.02, 0.03, 0.04, 0.06 and 0.08 mg a.s./kg soil (dry weight).

he following test parameters were assessed: behaviour,

mortality, metamorphosis (number of successful metamorphoses and time period to metamorphosis) and body weight of adult beetles.


X

5.3 Conclusion An LC50 of 0.046 mg a.s./kg soil (dry weight) was calculated (95% confidence limits: 0.036 - 0.062 mg/kg dry soil).

5.3.1 Reliability 1

5.3.2 Deficiencies None that affected the integrity of the findings.


Use separate "evaluation boxes" to provide transparency as to the comments and views submitted


Date



Conclusion

Reliability

Acceptability

Remarks

COMMENTS FROM ...



Date



Conclusion

Reliability

Acceptability

Remarks


Section A7.5.5.1 Annex Point IIA VII.7.5

Bioconcentration, further studies





-




Date


Conclusion

Remarks






Remarks



Section A7.5.5 Annex Point IIA VII.7.5

Bioconcentration, terrestrial





-




Date


Conclusion

Remarks






Remarks



Section A7.5.6 Annex Point IIIA XIII.3

Effects on other terrestrial non-target organisms





-




Date


Conclusion

Remarks






Remarks




Effect on mammals – acute oral toxicity





-




Date


Conclusion

Remarks






Remarks



Section A7.5.7.1.2 Annex Point IIIA XIII.3

Effect on mammals – short term toxicity





-




Date


Conclusion

Remarks






Remarks



Section A7.5.7.1.3 Annex Point IIIA XIII.3

Effect on mammals – effects on reproduction





-




Date


Conclusion

Remarks






Remarks


Section A7.1.1.1.1 Hydrolysis as a function of pH and...

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