1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Apr - 04 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4300 (Aerobic Aquatic Metabolism)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Draft SANCO 11802/2010/rev 7 in accordance with Regulation (EC) No 1107/2009

Deviations:

no

GLP compliance:

yes

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): The water and sediments were sampled freshly. Anglersee: Leverkusen/North Rhine-Westphalia/Germany; Wiehltalsperre Reichshof/North Rhine-Westphalia/Germany. Water and sediment were taken in approximately 0.4 to 0.5 m water depth and filled separately in plastic containers. Sediment was obtained from the upper sediment layer.
- Storage conditions: Stored at ambient conditions for three days until start of equilibration
- Storage length: 3 days
- Temperature at time of collection: Anglersee: 13 °C; Wiehltalsperre: 11 °C
- pH at time of collection: Anglersee: 7.8; Wiehltalsperre: 7.7
- Redox potential initial: Anglersee: 225 mv; Wiehltalsperre: 228 mv
- Oxygen concentrationl: Initial: Anglersee: @ 13 °C 10.52 mg/L; Wiehltalsperre: @ 11 °C 10.77 mg/l
TOTAL OC (mg/L): Anglersee: <2 / 2.4 / 14.7*; Wiehltalsperre: 2 / 6 / 87.8* *Measured at start of equilibration/DAT-0/DAT-101
- Water filtered: Yes
- Type and size of filter used: 0.063 mm mesh

Details on source and properties of sediment:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Anglersee, Leverkusen, Germany; Wiehltalsperre, Reichshof, Germany. Water and sediment were taken in approximately 0.4 to 0.5 m water depth and filled
separately in plastic containers. Sediment was obtained from the upper sediment layer.
- Storage conditions: Ambient conditions in the laboratory
- Storage length: 3 days until start of equilibration
- Textural classification: Anglersee Sand: 98, 2, 0 (Sand/SIlt/Clay%); Wiehltalsperre Silt Loam 36, 54, 10 (Sand/SIlt/Clay%)
- pH at time of collection: (sediment/0.01 M CaCl2 1/2) Anglersee 7.2; Wiehltalsperre 5.5; (sediment water 1/1) Anglersee 7.6; Wiehltalsperre 5.8
- Organic carbon: TOC (g/kg)* Anglersee 3.5/2.1/1.6; Wiehltalsperre 110/101/103 *measured at start of equilibration/DAT-0/DAT-101 (Days After Treatment [DAT])
- Redox potentiall: Initial: Anglersee 195 mv; Wiehltalsperre 66 mv
- CEC: Anglersee 2.7 meq/100 g; Wiehltalsperre 8.9 meq/100 g
Microbial activity: mg microbial CO2/hour/kg sediment dwt)* Anglersee 13.1/6.3/1.5/1.9**; Wiehltalsperre 117.9/129.2/53.8/54.6
*measured at start of equilibration/DAT-0/DAT-101 BIO-/BIO+
BIO- samples were left untreated
BIO+ samples were applied with solvent of application solution (400 μL methanol)
**Determination of microbial activity of system Anglersee was performed approximately 4 weeks
after the DAT-101 sampling interval.
- Sediment samples sieved: Yes

Duration of test (contact time):

101 d

Initial conc.:

99 µg/L

Based on:

act. ingr.

Remarks:

corresponding SAR of 51.5 μg per test system.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: Anglersee Sediment: (g wwt) 367.6, water (mL) 520; Wiehltalsperre Sediment: (g wwt) 215.4, water (mL) 520
- Solubilising agent (type and concentration if used): ACN evaporated to dryness followed by solubilization
- Test temperature: 20.3 °C
- pH: Anglersee: water mean 7.9, sediment mean 7.4; Wiehltalsperre water mean 7.5, sediment mean 6.8
- pH adjusted: no
- CEC: Anglersee: 2.7 meq/100 g; Wiehltalsperre 8.9 meq/100 g
- Continuous darkness: Yes
- Any indication of the test material adsorbing to the walls of the test apparatus: Mean material balances were 102.4% AR for system Anglersee (range from 101.1 to
103.7% AR) and 101.1% AR for system Wiehltalsperre (range from 98.5 to 102.3% AR).

TEST SYSTEM
- Culturing apparatus: Approximately 1000 mL cylindrical glass vessels inner diameter approx. 10.5 cm, surface area approx. 86.6 cm2
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: trap apparatus permeable to oxygen
- Method used to control oxygen conditions: cultured under ambient conditions
- Measuring equipment: Multi 350i

- Test performed in closed vessels due to significant volatility of test substance: The test substance and metabolites not volatile, however, system was clodsed using an oxygen permeable CO2 trap.
- Test performed in open system: No utilized an oxygen permeable CO2 trap.
- Details of trap for CO2 and volatile organics if used: Oxygen permeable trap consisted of in series a quartz/cotton wool plug, soda lime, quartz cotton wool plug, soda lime, quartz cotton woool plug and a polyurethane plug for absorption of volatile organic compounds.

SAMPLING
- Sampling frequency: Duplicate samples were processed and analyzed 0, 1, 3, 8, 14, 29, 59, and 101 days
after treatment (DAT).
- Sampling method used per analysis type: At each sampling interval, the water was separated from the sediment by decantation. The sediment was extracted twice at ambient temperature using acetonitrile/water 4/1 (v/v) and once using pure acetonitrile. Furthermore, two microwave-accelerated extraction steps were performed using acetonitrile/water 4/1 (v/v) at 70 °C and acetone/water 1/1 (v/v) at 50 °C. The amounts of test item and degradation products in water and sediment extracts were determined by liquid scintillation counting (LSC) and by HPLC/radiodetection analysis. The amount of volatiles and non-extractable residues were determined by LSC and combustion/LSC, respectively. Test item and degradation products were identified by HPLC-MS(/MS) including accurate mass determination, by 1H-NMR and/or by co-chromatography with reference items.

STATISTICAL METHODS: Nonlinear regression analysis was used to determine the kinetic parameters (KinGUI 2), and linear regression analysis was used to determine the radioactivity detector response (Microsoft® Excel).

Compartment:

natural water / sediment: freshwater

% Recovery:

102.4

Remarks on result:

other: Anglersee

Compartment:

natural water / sediment: freshwater

% Recovery:

101.1

Remarks on result:

other: Wiehltalsperre

Key result

Compartment:

natural water / sediment: freshwater

DT50:

11.1 d

St. dev.:

3.6

Type:

other: Double First Order Parallel

Temp.:

20.3 °C

Remarks on result:

other: Anglersee

Key result

Compartment:

natural water: freshwater

DT50:

5.3 d

St. dev.:

1.4

Type:

other: Double First Order Parallel

Temp.:

20.3 °C

Remarks on result:

other: Anglersee

Key result

Compartment:

natural water: freshwater

DT50:

6.3 d

St. dev.:

9.6

Type:

other: Double First Order in Parallel

Temp.:

20.3 °C

Remarks on result:

other: Wiehltalsperre

Key result

Compartment:

natural water / sediment: freshwater

DT50:

122 d

St. dev.:

0.8

Type:

other: Double First Order in Parallel

Temp.:

20.3 °C

Remarks on result:

other: Wiehltalsperre

Transformation products:

yes

No.:

#1

No.:

#2

Details on transformation products:

- Formation and decline of each transformation product during test: Test substance-N-methyl-quinazolinone and Test substance-N-methyl quinazolone-amide
- Pathways for transformation: Test substance to Test substance-N-methyl-quinazolinone to Test substance-N-methyl quinazolone-amide to Non-extractable residues (NER)

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study:Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: [Test substance-N-methyl-quinazolinone] was measured as 84.8% AR at DAT-59 (system Anglersee) and [ test substance-N-methyl-quinazolinone-amide] with 9.2% AR at DAT-101 (system Anglersee). [Test substance-N-methyl-quinazolinone] was measured as 32.6% AR at DAT-101 (system Wiehltalsperre) and [test substance-N-methyl-quinazolinone-amide] with 1.3% AR at DAT-101 (system Wiehltalsperre).
- Range of maximum concentrations in % of the applied amount at end of study period:[Test substance-N-methyl-quinazolinone] was measured as 80.4% AR at DAT-101 (system Anglersee) and [test substance-N-methyl-quinazolinone-amide] with 9.2% AR at DAT-101 (system Anglersee). [Test substance-N-methyl-quinazolinone] was measured as 32.6% AR at DAT-101 (system Wiehltalsperre) and [test substance-N-methyl-quinazolinone-amide] was measured as 1.3% AR at DAT-101 (system Wiehltalsperre).


MINOR TRANSFORMATION PRODUCTS
Not applicable

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
The total unidentified residues for both water/sediment systems amounted to a maximum of 4.1% AR and no single component exceeded 2.7% AR at any sampling interval for both water/sediment systems.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: System Anglersee: 101.1; System Wiehtalsperre: 101.3
- % of applied amount at end of study period: System Anglersee: 94.6; System Wiehtalsperre: 90.0

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: System Anglersee: 0.2; System Wiehtalsperre: 0.3
- % of applied amount at end of study period: System Anglersee: 7.7; System Wiehtalsperre: 10.3

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 0.2% at DAT-101

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

Summary of the Kinetic Evaluation (for Trigger Values According to FOCUS) of the Degradation of Test Substance in Water/Sediment Systems under Aerobic Conditions

Water/sediment system	Compartment	Kinetic	DT50	DT90	chi^2error	Visual
(Sediment Type)		Model^1	[d]	[d]	[%]	Assessment ^2
Anglersee (sand)	Water	DFOP	5.3	26.6	1.4	+
	Entire System	SFO	11.1	37.0	3.1	+
Wiehltalsperre (silt loam)	Water	DFOP	6.3	27.8	9.6	+
	Entire System	DFOP	122	> 1000	0.8	+
^1SFO: Single first order, DFOP = double first order in parallel
^2Visual assessment: + good

Reference 2

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sep 2014 - 21 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OCSPP Guideline Number: 835.SUPP

Version / remarks:

Alternatively: USEPA OPPTS 835.3190 (Aerobic Mineralization in Surface Water - Simulation Biodegradation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 283/2013 in accordance with Regulation (EC) No 1107/2009

Deviations:

no

GLP compliance:

yes

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Location: Reichshof, North Rhine-Westphalia, Germany
Sampling Depth: 20 - 50 cm
Contamination history: Reservoir used for the preparation of drinking water.
- Storage conditions: Stored until filtration at 5 °C at the receiving facility and was directly processed after filtration.
- Storage length: 1 day until filtration and equilibration to study conditions/sterilization.
- Temperature at time of collection: 9.7 °C
- pH at time of collection: 8.0
- Redox potential (mv) initial/final: Low Concentration: 185/167 mean: 172; High Concentration: 186/168 mean: 169
- Oxygen concentration (mg/l) initial/final: Low Concentration: 7.99/8.41 mean: 8.1; High Concentration: 8.45/8.25 mean: 8.2
- Dissolved organic carbon (%): < 2.0 mg/L
- Microbial activity of the surface water was demonstrated by extensive mineralization (formation of carbon dioxide of 41.2% AR for BIO- and 35.5% AR for BIO+ after 3 days) of the control item benzoic acid. The amount of control item decreased to below detection limit after three days of incubation confirming that the microbial activity of the surface water was sufficient for the test.
- Water filtered: Yes
- Type and size of filter used: 0.063 mm filter

Duration of test (contact time):

>= 63 - <= 65 d

Initial conc.:

10.7 µg/L

Based on:

act. ingr.

Initial conc.:

53.7 µg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 100 mL
- Composition of medium: Fresh surface water
- Solubilising agent (type and concentration if used): Methanol
- Test temperature: 20+/- 2 °C
- pH: Low concentration mean: 9.4; High concentration mean: 9.4
- pH adjusted: yes/no No
- Aeration of dilution water: No
- Continuous darkness: Yes
- Any indication of the test material adsorbing to the walls of the test apparatus: No

TEST SYSTEM
- Culturing apparatus: 250 mL Erlenmeyer glass flasks with baffles
- Number of culture flasks/concentration: duplicate vessels for each sampling interval
- Method used to create aerobic conditions: No methods were required to creat aeroboc conditions. Upon collection the water was characterized as having an oxygen saturation percentage of 84%
- Method used to control oxygen conditions: trap attachments were permeable for oxygen

- Test performed in closed vessels due to significant volatility of test substance: Test was performed with volatile traps permeable to oxygen.
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: Each test vessel was fitted with a trap attachment (permeable for oxygen)
containing soda lime for absorption of carbon dioxide and a polyurethane (PU) foam plug for adsorption of volatile organic compounds (VOC).

SAMPLING
- Sampling frequency: Twelve sampling intervals were distributed over the entire incubation period of 63 days. Duplicate samples of each test concentration were processed and analyzed 0, 0.08, 0.16, 0.25, 1, 3, 7, 14, 21, 30, 45 and 63 days after treatment (DAT). Sterile samples were processed at DAT-65 for both concentrations. Microbial activity samples (BIO-) were processed in duplicate at start of the study and 3 days after treatment. Solvent control microbial activity samples (BIO+) were processed 3 days after treatment.
- Sterility check if applicable: At DAT-65
- Sample storage before analysis: The trap attachments containing soda lime and PU foam were stored before processing
at ambient temperature in the laboratory for a maximum period of 1 day. The first analyses of surface water by LSC and the primary chromatographic method were usually done within three days after sampling. After analysis, samples were stored at < -18 °C in the dark.
- Other: Due to high radioactivity at the origin, samples of DAT-1 were re-analyzed after a sample storage period was 31 days.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Sterile samples were prepared to determine influences on the degradation of the test item induced by hydrolysis (4 samples per concentration). Therefore, the respective test systems were sterilized by autoclaving for 90 minutes at 121 °C. Trap attachments were autoclaved separately. The sterilized test systems were stored under a clean bench until to application. Sterile surface water samples were autoclaved once more prior to application.
- Other: benzoic acid was utilized as a + control

STATISTICAL METHODS: Not applicable

Reference substance:

benzoic acid, sodium salt

Compartment:

natural water: freshwater

% Recovery:

105.7

Remarks on result:

other:

Remarks:

Low concentration (10.7 µg/L)

Compartment:

natural water: freshwater

% Recovery:

104

Remarks on result:

other:

Remarks:

High concentration (53.7 µg/L)

Key result

Compartment:

natural water: freshwater

DT50:

0.9 d

St. dev.:

5.3

Type:

other: First Order Multi Compartment

Temp.:

20.5 °C

Remarks on result:

other:

Remarks:

Low concentration (10.7 µg/L)

Key result

Compartment:

natural water: freshwater

DT50:

0.9 d

St. dev.:

5.6

Type:

other: First Order Multi Compartment

Temp.:

20.5 °C

Remarks on result:

other:

Remarks:

High Concentration (53.7 µg/L)

Transformation products:

yes

No.:

#1

Details on transformation products:

- Formation and decline of each transformation product during test: test substance-N-methyl-quinazolinone was identified by TLC co-chromatography as well
as by HPLC-MS(/MS) including accurate mass determination after isolation from surface water samples
- Pathways for transformation: test substance-N-methyl-quinazolinone was formed via direct transformation from test substance under the conditions of the study.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: test substance-N-methylquinazolinone was formed with 104.2% AR at DAT-45 in low concentration samples and 100.5% AR at DAT-45 in high concentration samples. The total unidentified residues amounted to a maximum of 5.7% AR and no single component exceeded 4.3% AR at any sampling interval for both concentrations.
- Range of maximum concentrations in % of the applied amount at end of study period: Low test concentration 97% +/- 0.5% AR at DAT-63; High test concentration 92.1% +/1 0.3% AR at DAT-63

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: The total unidentified residues amounted to a
maximum of 5.7% AR and no single component exceeded 4.3% AR at any sampling interval for both concentrations.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Carbon dioxide accounted for a maximum amount of 1.4% AR on DAT-14 abd subsequently declined to < 0.1% at DAT63 in the low concentration treatment group. In the high concentration treatment group CO2 ws measured as < 0.1% for the entire study.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Formation of VOC was insignificant as demonstrated by values of ≤ 2.3% AR at all sampling intervals for both concentrations.

STERILE TREATMENTS (if used)
- Transformation of the parent compound: In sterile samples (DAT-65), the amount of test substance in surface water was 3.6% +/- 0% AR in low concentration test systems and 3.5% +/- 0.1% AR in high concentration test systems.
- Formation of transformation products: In sterile samples, the amount of test substance-N-methyl-quinazolinone in the surface
water at DAT-65 was 96.8% +/- 1.2% AR for low concentration samples and 94.6% +/- 0.3% AR for high concentration samples.
- Volatilization: In sterile samples (DAT-65), the amount of volatile compounds in surface water was < 1.1% +/- 0% AR in the low concentration test systems and 0.5% +/- 0.2% AR in high concentration test systems.

Results with reference substance:

Microbial activity of the surface water was demonstrated by extensive mineralization (formation of carbon dioxide of 41.2% AR for BIO- (microbiol activity control) and 35.5% AR for BIO+ (solvent control microbiol activity control) after 3 days) of the control item benzoic acid. The amount of control item decreased to below detection limit after three days of incubation confirming that the microbial activity of the surface water was sufficient for the test.

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jan 2014 - 21 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4400 (Anaerobic Aquatic Metabolism)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 283/2013 / DRAFT SANCO 11802/2010/rev 7 in accordance with Regulation (EC) No 1107/2009

Deviations:

no

GLP compliance:

yes

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Radiolabelling:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
North Carolina (NC): Water: Submersion (approximately 40 cm deep) of closed plastic container, then opening and allowing to fill.
California (CA): Collected from overlying pond water.
Contamination history: NC No pesticide used in 17 years; CA No pesticide used

- Storage conditions: Ambient conditions during shipment and then stored at <5.5 ºC until test
system preparation
- Storage length: NC system: 10-days to preincubation, CA system 5-days to preincubation
- pH at time of collection: NC 6.9; CA 9.0 both at time of characterization
- Electrical conductivity: NC 0.10; CA 0.30 [mmhos/cm]
- Redox potential (mv) initial/final: NC -128.5/-1.9; CA 54.5/33.6
- Oxygen concentration (mg/l) initial/final: NC 0.3/0.1; CA 0.3/0.1
- Hardness (CaCO3): [mg/L] NC 19; CA 102
- Dissolved organic carbon: [mg/L] NC 4.8; CA 3.1
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Initial/Final [mg/g] NC 2.4E01/0.649; CA 3.99E01/2.19E01
- Water filtered: Yes
- Type and size of filter used: Water and sediment were sieved with a 2-mm sieve

Details on source and properties of sediment:

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
NC: Careful removal of the top 10 cm of sediment with a flat shovel; CA Collected from the top 5-10 cm of pond sediment with shovel.
Contamination history: NC No pesticide used in 17 years; CA No pesticide used
- Storage conditions: Ambient conditions during shipment and then stored at <5.5 ºC until test
system preparation
- Storage length: NC system: 10-days to preincubation, CA system 5-days to preincubation
- Textural classification (i.e. %sand/silt/clay): NC 45.9/29.524.6; CA 87.7/5.0/7.3
- pH at time of collection: [0.01 M CaCl2] NC: 4.6; CA: 7.7
- Organic carbon (%): NC: 1.8; CA: 0.33
- Redox potential (mv) initial/final: NC -80.2/29.3; CA -135.7/-21.7
- CEC (meq/100 g): NC: 6.5; CA: 5.0
- Bulk density (g/cm³): NC 1.11; CA 1.32
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Initial/Final NC: 2.4E03/6.5E01; CA 4.0E03/2.2E03
- Sediment samples sieved: yes/no Yes

Duration of test (contact time):

104 d

Initial conc.:

98 other: micrograms/test system

Based on:

act. ingr.

Remarks:

Kinetics analysis

Initial conc.:

210 other: micrograms/test system

Based on:

act. ingr.

Remarks:

Metabolite ID

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 60 g dwt sediment/180 ml water
- Solubilising agent (type and concentration if used): methanol (<0.1%)
- Test temperature: 20.1 +/- 0.1 °C
- pH: NC: 5.2; CA 8.0
- pH adjusted: No
- CEC (meq/100 g): NC: 6.5; CA 5.0
- Aeration of dilution water: No
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: No, Overall recoveries showed that there was no radioactivity loss. The average recovery was 99.8% for NC and 98.2% for CA (n = 8).

TEST SYSTEM
- Culturing apparatus: 250 mL Pyrex side arm Erlenmeyer flasks
- Number of culture flasks/concentration: Twenty-four test systems were treated at the kinetics rate, four test systems were treated at two times the kinetics rate for metabolite identification purposes, two test systems were treated only with solvent and six were left untreated.:
- Method used to create anaerobic conditions: For each water/sediment system, test systems were set up and pre-equilibrated to achieve anaerobic condition. During the pre-equilibration period, each test system was equipped with mineral oil traps, flushed with nitrogen, and acclimated in a nitrogen filled incubator.
- After treatment of test systems, double-valved tops were placed on each flask
- Measuring equipment: Radiometric measurements were made using a Tri-Carb model B29100 (Perkin Elmer, Waltham, MA, USA) liquid scintillation counter equipped with automatic external standardization for quench correction. Liquid samples were radioassayed using Ultima Gold™ liquid scintillation cocktail. Solid samples (after extraction) were oxidized using a Model OX700 or OX500 (R.J. Harvey Instrument Corp., Hillsdale, NJ, USA) sample oxidizer. The generated 14CO2 was radioassayed with 15 mL of Harvey Carbon-14 oxidizer cocktail. The samples were radioassayed for 14Ccontent by LSC, and the results were corrected for oxidizer efficiency

- Test performed in closed vessels due to significant volatility of test substance: The test system was closed although significant volatility of the test substance was not anticipated.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Test systems were closed during incubation and volatiles were trapped for the replicates taken at each sampling interval by flushing the system with nitrogen. Volatile traps were comprised of five 50-mL tubes consisting of (1) blank (2) 30 mL of ethylene glycol (3 & 4) 30 mL of 2.0 N KOH (potassium hydroxide) (5) 30 mL of 1.0 M sulfuric acid. Samples were purged prior to extraction with nitrogen for 10 minutes into volatile trapping solutions. The water was then separated from the sediment by decanting and filtering.

SAMPLING
- Sampling frequency: Eight sampling intervals were conducted over a period of 104 days at 0, 6, 12, 20, 40, 60, 82, and 104 days post treatment.
- Sampling method used per analysis type:
Aqueous: For both sediment/water systems, the aqueous portions of test systems were decanted into a 250-mL Teflon® centrifuge bottle, centrifuged for 10 minutes at 1,700 g, and the supernatant then decanted into a 250-mL graduated cylinder. Triplicate 0.5-mL aliquots of the sample were radioassayed with 5 mL Ultima Gold™ liquid scintillation cocktail, and a separate aliquot was transferred to an HPLC autosample vial for analysis by HPLC.

Ambient sediment extraction: The sediment portion of the sample was transferred into the same 250 mL Teflon® centrifuge bottle that had contained the aqueous phase sample. The sediment was extracted with approximately 80 mL of ACN:H2O 4:1 (v:v) and extracted for 20 minutes by shaking on a bench-top shaker at ambient temperature. The sample was centrifuged for 10 min at 1,700 g, and the supernatant was decanted into a 500 mL graduated cylinder through a Whatman GF/F filter. The sediment was extracted one more time with 80 mL of ACN:water 4:1 (v:v) and again with acetonitrile, the supernatant from each extract was filtered through the same filter into the graduated cylinder, and the volume was recorded. Triplicate 0.5-mL aliquots of the ambient extract were radioassayed with 5 mL Ultima Gold™. An aliquot of both the ambient extract and microwave extracts (approximately 10-20% each of the total extract volume) was combined and rotary evaporated under vacuum at approximately 30 °C before HPLC analysis. The volume concentrated typically allowed for detection of approximately <2% of the applied radioactivity and 2 injections for each sample. Concentration recoveries were determined on representative samples to determine if loss of radioactivity occurred during processing.

Microwave sediment extraction: The sediment after extraction at ambient temperature was extracted with 80 mL of ACN:water 4:1 (v:v), stirring in the microwave for 10 minutes at 70 °C and then centrifuged for 10 min at 1,700 g. The supernatant was filtered through the same filter used for ambient extraction into a 250 mL graduated cylinder. The sediment was then extracted a second time with 80 mL acetone using the microwave for 10 minutes at 50 oC. The supernatant was then filtered through the same Whatman GF/F filter as above into the 250 mL graduated cylinder and the total volume was recorded. Triplicate 0.5 mL aliquots of the microwave extract were radioassayed with 5 mL Ultima Gold™. An aliquot of the microwave extract was combined with an aliquot of the ambient extract as described above and
concentrated prior to HPLC analysis.

Sediment cake: The extracted sediment samples were air dried, weighed and homogenized thoroughly with coffee grinder. Triplicate aliquots (0.5 to 1.0 g) of the sediment were analyzed by combustion.

- Sample storage before analysis: Samples were processed and the sediment was extracted on the day of sampling. The extract samples and water samples were initially analyzed within 6 days of the interval, thus there was no need for storage stability data.

Compartment:

natural water / sediment: freshwater

% Recovery:

99.8

St. dev.:

2.5

Remarks on result:

other: North Carolina

Compartment:

natural water / sediment: freshwater

% Recovery:

98.2

St. dev.:

3.2

Remarks on result:

other: California

Key result

Compartment:

natural water: freshwater

DT50:

16 d

St. dev.:

1.3

Type:

other: Double First Order in Parallel

Temp.:

20.1 °C

Remarks on result:

other: North Carolina

Key result

Compartment:

natural water / sediment: freshwater

DT50:

217.9 d

St. dev.:

1.947

Type:

other: Double First Order in Parallel

Temp.:

20.1 °C

Remarks on result:

other: North Carolina

Key result

Compartment:

natural water: freshwater

DT50:

24.1 d

St. dev.:

3.4

Type:

other: Double First Order in Parallel

Temp.:

20.1 °C

Remarks on result:

other: California

Key result

Compartment:

natural water / sediment: freshwater

DT50:

103.5 d

St. dev.:

2.945

Type:

other: Double First Order in Parallel

Temp.:

20.1 °C

Remarks on result:

other: California

Mineralization rate (in CO2):

0.5 other:

Transformation products:

yes

No.:

#1

Details on transformation products:

- Pathways for transformation: Test substance to N-methyl-quinazolinonen: Test substance degrades in anaerobic water/sediment systems to form one major
degradate, which was characterized in the study. N-methyl-quinazolinone maintains all the ring structures of the parent compound. This indicates that this study with one radiolabel position is sufficient to determine the complete degradation pathway of the test substance.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Remarks:

In the NC test systems NER increased from below the limit of detection at Day 0 to 10.4% at Day 82 then decreased to 9.4 at Day 104. In the CA systems NER increased from below the limit of detection at Day 0 to 4.9% at the end of the study, Day 104.

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
In the NC water phase N-methylquinazolinone was detected at 1.0% of the applied radioactivity at Days 12 and 20 and decreased to below the limit of detection at Day 40 where it stayed for the remainder of the study. In the NC sediment phase N-methyl-quinazolinone formed at 4.3% of the applied radioactivity at Day 6 and increased to 19.5% by Day 104. In the NC total water/sediment system N-methyl-quinazolinone formed at 4.3% of the applied radioactivity at Day 6, reached a maximum level of 19.5% in the total system at Day 104. No other degradates were detected in the NC test systems.
In the CA water phase N-methylquinazolinone was detected in the water phase at a maximum level of 6.1% at Day 20 and decreased to 2.9% by the end of the study. In the CA sediment phase, N-methyl-quinazolinone formed at 0.3% of the applied radioactivity at Day 0 and increased to 41.1% at Day 104. In the total CA water/sediment system N-methyl-quinazolinone formed at 3.5% of the applied radioactivity at Day 0 and increased to 44.0% by Day 104. Individual unidentified minor degradates were detected but did not exceed a mean of 2.7% of the applied radioactivity.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: In the NC total water/sediment systems no other degradates were formed in addition to N-methyl-quinazolinone. In the CA total water/sediment systems additional unidentified minor degradates were detected but did not exceed a mean of 2.7% of the applied radioactivity.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: In the CA total water/sediment systems additional unidentified minor degradates were detected but did not exceed a mean of 2.7% of the applied radioactivity.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: NC water phase 91%, sediment phase 5.2%; CA water phase 90.7%, sediment phase 6.3%
- % of applied amount at end of study period: NC water phase 12.1%, sediment phase 78.1%; CA water phase 27.3%, sediment phase 61.6%

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: NC < LOD; CA < LOD
- % of applied amount at end of study period: NC 10.4% on Day 82 and decreased to 9.4% at the end of the study (Day 104). CA 4.9% at the end of the study (Day 104).

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: NC at Day-104 [14C] CO2 remained at ≤0.5% of the applied radioactivity. CA at Day-104 [14C] CO2 remained at ≤0.1% of the applied radioactivity.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: NC Volatile compounds remained low throughout the study at ≤0.4% AR.; CA Volatiles remained ≤0.1% throughout the study.

Water/sediment system	Compartment	Kinetic	DT50	DT90	chi^2error
(Sediment Type)		Model^1	[d]	[d]	[%]
NC (loam)	Water	DFOP	16.0	213.4	1.3
	Entire System	DFOP	217.9	723.7	1.947
CA (loamy sand)	Water	DFOP	24.1	252.4	3.4
	Entire System	DFOP	103.5	363.1	2.945

Description of key information

Aerobic Aquatic Water/Sediment Systems (OECD 308):

DT50 water: 5.3 - 6.3 days at 20.3 °C , M-492584-01-1

DT50 water/sediment: 11.1 - 122 days at 20.3 °C , M-492584-01-1

 

Anaerobic Aquatic Water/Sediment Systems: (OECD 308):

DT50 water: 16 - 24.1 days at 20.1 °C, M-546847-01-2

DT50 water/sediment: 104 - 218 days at 20.1 °C, M-546847-01-2

 

Aerobic Aquatic Mineralization in Surface Water (OECD 309):

DT50 water: 0.9 days at 20.5 °C, M-545815-01-2

Key value for chemical safety assessment

Half-life in freshwater:

6.3 d

at the temperature of:

20.3 °C

Half-life in freshwater sediment:

122 d

at the temperature of:

20.3 °C

Additional information

Aerobic Aquatic Water/Sediment Systems:

The route and rate of degradation were investigated in two water/sediment systems (source: Anglersee and Wiehltalsperre) under aerobic conditions in the dark in the laboratory for 101 days at 20.3 °C (M-492584-01-1). The study followed the OECD Guideline for the Testing of Chemicals No. 308. The study application rate was 51.5 µg/test system, corresponding to 99.0 µg/L. Duplicate test systems were processed and analyzed 0, 1, 3, 8, 14, 29, 59 and 101 days after treatment.

Overall mean material balance was 102.4% of applied radioactivity (%AR) for water/sediment system Anglersee and 101.1%AR for water/sediment system Wiehltalsperre. The maximum amount of carbon dioxide produced was 0.2%AR at study end (DAT‑101) in both water/sediment systems. Formation of volatile organic compounds (VOC) was insignificant as demonstrated by values of ≤ 0.1%AR at all sampling intervals for both water/sediment systems.

Residues in water decreased from 93.7 to 7.2%AR in system Anglersee and from 92.5 to 5.1%AR in system Wiehltalsperre from DAT-0 to DAT-101. Extractable residues in sediment increased from 7.4 to 87.4%AR in system Anglersee and from 8.8 to 84.9%AR in system Wiehltalsperre from DAT‑0 to DAT‑101. Extractable residues in the total system (water and sediment extracts) decreased in system Anglersee from 101.1 to 94.6%AR from DAT-0 to DAT-101. In system Wiehltalsperre, extractable residues in the total system decreased from 101.3 to 87.9%AR from DAT-0 to DAT-59 and increased then slightly to 90.0%AR at DAT-101.

Non-extractable residues (NER) increased in system Anglersee from 0.2 to 7.7%AR from DAT‑0 to DAT‑101. In system Wiehltalsperre, NER increased from 0.3 to 12.1%AR from DAT-0 to DAT-29 and decreased then slightly to 10.3%AR at DAT-101.

The test substance dissipated from the water due to degradation and translocation into the sediment. The amount of test substance in the water in system Anglersee was 92.9%AR at DAT-0 and was not detectable at DAT-101. In system Wiehltalsperre, the amount of test substance in the water decreased from DAT‑0 to DAT‑101 from 92.5 to 4.3%AR. The amount of test substance in the sediment extracts increased in system Anglersee from DAT-0 to DAT-8 from 7.4 to 22.1%AR and decreased then to below limit of detection (LOD) at DAT‑101. In system Wiehltalsperre, the amount of test substance in the sediment extracts increased from 8.8%AR at DAT‑0 to 64.1%AR at DAT-14 and decreased then to 47.7%AR at DAT-101.

The amount of test substance in the total system decreased from 100.2%AR to < LOD in system Anglersee and from 101.3 to 52.0%AR in system Wiehltalsperre from DAT-0 to DAT-101.

Two degradation products were identified with the following maximum occurrences in the total system: [test substance]-N methyl-quinazolinone with 84.8%AR at DAT-59 (system Anglersee) and [test substance]-N methyl-quinazolinone-amide with 9.2%AR at DAT-101 (system Anglersee). The total unidentified residues for both water/sediment systems amounted to a maximum of 4.1%AR and no single component exceeded 2.7%AR at any sampling interval for both water/sediment systems.

The experimental data could be best described by single first order (SFO) and double first order in parallel (DFOP) kinetic models. The DT50 values for the dissipation of the test substance from the water were 5.3 and 6.3 days in system Anglersee and Wiehltalsperre, respectively. The DT50 values for the degradation of the test substance in the total water/sediment system were 11.1 and 122 days in system Anglersee and Wiehltalsperre, respectively.

It is concluded that under aerobic conditions the test substance and its degradation products will be degraded in the aquatic environment.

 

Anaerobic Aquatic Water/Sediment Systems:

The route and rate of degradation were investigated in two water/sediment systems (source: North Carolina and California) under anaerobic conditions in the dark in the laboratory for 104 days at 20 °C (M-546847-01-2). The study followed the OECD Guideline for the Testing of Chemicals No. 308. The study application rate was 17.6 µg/test system, corresponding to 100 µg/L. Duplicate test systems were processed and analyzed 0, 6, 12, 20, 40, 60, 82 and 104 days after treatment.

Overall mean material balance was 99.8% of applied radioactivity (%AR) for water/sediment system North Carolina (NC) and 98.2%AR for water/sediment system California (CA). Volatile compounds (carbon dioxide and volatile organic compounds) remained low throughout the study at ≤0.4%AR for water/sediment system NC and at ≤0.1%AR for water/sediment system CA.

Residues in water decreased from 91.0%AR at DAT-0 to 12.1%AR at DAT-104 in water/sediment system NC and from 90.7%AR at DAT-0 to 27.3%AR at DAT-104 in water/sediment system CA. Extractable residues in sediment increased from 5.2%AR at DAT-0 to 78.1%AR at DAT-104 in water/sediment system NC and from 6.3%AR at DAT-0 to 61.6% at DAT-82 and decreased to 61.0%AR at DAT-104 in water/sediment system CA.

Non-extractable residues (NER) increased from below the detection limit at DAT-0 to 10.4%AR at DAT‑82 and slightly decreased to 9.4%AR at DAT-104 in water/sediment system NC. In water/sediment system CA NER increased from below the detection limit at DAT-0 to 4.9%AR at DAT-104.

The Test substance dissipated from the water due to transformation and translocation into the sediment. The amount of test substance in the water decreased from DAT-0 to DAT-104 from 91.0 to 12.1%AR in water/sediment system NC and from 87.5 to 24.4%AR in water/sediment system CA.

The amount of the test substance in the sediment extracts increased in water/sediment system NC from DAT-0 to DAT-60 from 5.0 to 67.0%AR and decreased then to 58.6%AR at DAT-104. In water/sediment system CA the amount of the test substance in the sediment extracts increased from DAT-0 to DAT-12 from 5.9 to 30.0%AR and decreased then to 19.9%AR at DAT-104.

The amount of the test substance in the total system decreased from DAT-0 to DAT-104 from 96.0 to 70.7%AR in water/sediment system NC and from 93.3 to 44.3%AR in water/sediment system CA.

One major degradation product, [test substance]-N-methyl-quinazolinone, was identified during the study with the following maximum amounts: max. water: 6.1%AR at DAT-20; max. sediment: 41.1%AR at DAT-104; max. entire system: 44.0%AR at DAT-104.

The experimental data could be well described by a double first order in parallel (DFOP) kinetic model. The DT50 values for the dissipation of the test substance from the water were 16.0 and 24.1 days in water/sediment system NC and CA, respectively. The DT50 values for the degradation of the test substance in the total water/sediment system were 218 and 104 days in days in water/sediment system NC and CA, respectively.

It is concluded that the test substance will be degraded slowly in an anaerobic aquatic water/sediment environment.

 

Aerobic Aquatic Mineralization in Surface Water:

The route and rate of degradation were studied at two test concentrations in surface water under aerobic conditions (pelagic test) in the laboratory in the dark at 20 ± 2 °C for 65 days at maximum (M-545815-01-2). The study followed the OECD Guideline for the Testing of Chemicals No. 309. Study application rates of 10.7 μg/L and 53.7 μg/L surface water were applied for low and high concentration samples, respectively. The test was performed in test systems consisting of Erlenmeyer flasks with baffles each containing 100 mL of the sampled surface water and equipped with traps (permeable for oxygen) for the collection of carbon dioxide and volatile organic compounds. The surface water in the test systems was kept in motion during the entire study period. Duplicate samples of each test concentration were processed and analyzed 0, 0.08, 0.16, 0.25, 1, 3, 7, 14, 21, 30, 45 and 63 days after treatment (DAT). Sterile samples of both concentrations were processed and analyzed at DAT-65. The amounts of test item and degradation products in surface water were determined by liquid scintillation counting (LSC) and by TLC/ radiodetection analysis. The amount of volatiles was determined by LSC. Test item and degradation products were identified by HPLCMS(/MS) including accurate mass determination and/or by co-chromatography with reference items. Mean material balances were 104.5% AR for low concentration test systems (range from 100.8 to 107.1% AR) and 102.7% AR for high concentration test systems (range from 99.9 to 104.4% AR).

The maximum amounts of carbon dioxide were ≤ 1.4% AR at all sampling intervals for both concentrations. The amounts of carbon dioxide formed in sterile samples after 65 days were 0.3% AR for both concentrations. Formation of VOC was insignificant as demonstrated by values of ≤ 2.3% AR at all sampling intervals for both concentrations in degradation samples as well as in sterile samples.

The residues in surface water ranged between 100.6 and 107.1% AR in low concentration test systems and between 99.9 and 104.3% AR in high concentration test systems for all sampling intervals. In sterile samples, residues in surface water amounted to 105.5% AR in low concentration test systems and 103.4% AR in high concentration test systems.

The amount of test substance in surface water decreased from DAT-0 to DAT-63 from 97.2% AR to < LOD in low concentration test systems and from 93.5 to 3.7% AR in high concentration test systems. In sterile samples (DAT-65), the amount of test substance in surface water was 3.6% AR low concentration test systems and 3.5% AR in high concentration test systems.

One degradation product was identified with the following maximum occurrences: [test substance]-N-methyl-quinazolinone with 104.2% AR at DAT-45 in low concentration samples and 100.5% AR at DAT-45 in high concentration samples. The total unidentified residues amounted to a maximum of 5.7% AR and no single component exceeded 4.3% AR at any sampling interval for both concentrations.

In sterile samples, the amount of [test substance]-N-methyl-quinazolinone in the surface water at DAT-65 was 96.8% AR for low concentration samples and 94.6% AR for high concentration samples. The experimental data could be best described by a first order multi compartment (FOMC) kinetic model. The DT50 values for the test substance in the tested surface water under aerobic conditions were 0.9 days for both concentrations.

Formation of carbon dioxide was insignificant during the test, indicating that mineralization plays only a minor role in the fate of the test substance in surface water under the conditions of the test.