4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Mar - 10 Nov 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Environmental Agency of Japan on July 01, 1978: Guideline for the Experiment of Pesticide Residue in Soil

Qualifier:

according to guideline

Guideline:

other: 59 NOSAN No. 4200 (MAFF Jan. 28,1985)

GLP compliance:

yes

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: According to USDA system, International system, and Japan agricultural society system

Remarks:

see Table "Soil properties" below

Soil no.:

#1

Soil type:

other: clay loam (USDA system)

% Clay:

30.9

% Silt:

41.2

% Sand:

28

% Org. C:

2.4

pH:

6.7

CEC:

21.5 meq/100 g soil d.w.

Soil no.:

#2

Soil type:

other: silty loam (USDA system)

% Clay:

41.1

% Silt:

46.3

% Sand:

12.6

% Org. C:

6.32

pH:

6.4

CEC:

36.2 meq/100 g soil d.w.

Soil no.:

#1

Soil type:

other: clay loam (International system)

% Clay:

18.5

% Silt:

25.8

% Sand:

55.7

% Org. C:

2.4

pH:

6.7

CEC:

21.5 meq/100 g soil d.w.

Soil no.:

#2

Soil type:

other: Light clay (International system)

% Clay:

29.1

% Silt:

29.8

% Sand:

41.1

% Org. C:

6.32

pH:

6.4

CEC:

36.2 meq/100 g soil d.w.

Soil no.:

#1

Soil type:

other: Clay loam (Japan agricultural society system)

% Clay:

23.2

% Silt:

29.7

% Sand:

47.1

% Org. C:

2.4

pH:

6.7

CEC:

21.5 meq/100 g soil d.w.

Soil no.:

#2

Soil type:

other: Loam (Japan agricultural society system)

% Clay:

7.5

% Silt:

42.7

% Sand:

49.8

% Org. C:

6.32

pH:

6.4

CEC:

36.2 meq/100 g soil d.w.

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location:
soil #1: JAPR Ryugasaki, Japan (soil genesis: alluvial, low organic Carbon)
soil #2: JAPR Ushiku, Japan (soil genesis: volcanic ash, high organic carbon)
- Collection procedures: Surface soil was collected
- Storage conditions: The soils were stored under aerobic conditions in plastic bags at +4 °C in a refrigerator prior to the beginning of the study.
- Soil preparation (e.g., 2 mm sieved; air dried etc.): Before the start of the experiment, the soils were gently air-dried so that they could be sieved to <2 mm.

PROPERTIES OF THE SOILS (in addition to defined fields)
- Dry substance (DS) of test soil used (%): soil #1: 81.06; soil #2: 67.88
- Soil density (g/cm³): 1
- Required amount of water for getting a water layer of 1.5 cm above the soil: soil #1:119 mL; soil #2: 125 mL

Soil No.:

#1

Duration:

105 d

Soil No.:

#2

Duration:

105 d

Soil No.:

#1

Initial conc.:

0.28 kg/ha d.w.

Based on:

act. ingr.

Soil No.:

#2

Initial conc.:

0.28 kg/ha d.w.

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

28.1 ± <1 °C

Soil No.:

#2

Temp.:

28.1 ± <1 °C

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): The batches with the submerged soils were prepared on March 7 and 8, 1995 and pre-incubated under the projected study conditions at 28 ± 1 °C in the dark until March 21, 1995.
- Soil condition: air dried
- Soil (g/replicate): 50 g dw
- No. of replication treatments: 3
- Test apparatus (Type/material/volume): All the vessels intended for processing at interval day 0 (including the Bio vessels) were closed with a plug of cotton wool for short term, only. During pre-incubation all vessels were closed in the same manner.
- Details of traps for CO2 and organic volatile, if any: The trap attachment is permeable for oxygen, however, allows to absorb CO2 by soda lime and volatile metabolites by polyurethane foam. At the time of processing (at each sampling interval) and before taking off the trap attachment, gaseous compounds contained in the test vessel were purged into the trap by air for about 1 minute.

Test material application
- Volume of test solution used/treatment: The dosed amount of active ingredient was calculated according to an expected recommended maximum dose rate of about 300 g test substance per hectare. Considering a soil depth of 10 cm and a soil density of 1 g/cm3 this corresponds to a weighed amount of 15 ug test substance per 50 g DS of soil (i.e. per incubation batch) or to 0.3 mg/kg of soil.
- Application method (e.g. applied on surface, homogeneous mixing etc.): After determination of the content of dry substance, each 50 g DS was weighed into a 300 mL Erlenmeyer flask. Then the soil was flodded by adding purified water (deionized water) in order to get a water layer of about 1.5 cm on top of the soil surface. The exact volume of dosed water/vessel was determined in a pre-test and was constant for all the vessels containing the same soil. The depth of soil layer was about 13 mm and 19 mm in the submerged alluvial and volcanic ash soil, respectively.

Any indication of the test material adsorbing to the walls of the test apparatus: no

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: At the start of incubation the depth of supernatant water in the test systems was about 1.5 cm. Relevant losses of water out of the system (i.e. of testing group #1) were not observed during the course of study.
- Continuous darkness: Yes
- Incubation conditions: The vessels were placed on a shaking platform and were shaken slowly (about 50 rpm) in order to avoid a disturbance of soil but to guarantee an oxygen exchange with the water (simulation of movement of water by a blowing wind over a paddy soil field or by prevailing temperature gradients).


OXYGEN CONDITIONS (delete elements as appropriate)
- The content of dissolved oxygen (DO), the pH value and the redox potential (RP) were measured in the surface water as well as pH and RP in the submerged soil by a suitable electrode.
- Methods used to create the an/aerobic conditions: see details for "Microbial activity, pH-value, redox potential and oxygen content" under "Any other information on results incl. tables" below
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): see details for "Microbial activity, pH-value, redox potential and oxygen content" under "Any other information on results incl. tables" below

SAMPLING DETAILS
- Sampling intervals: On days 0, 3, 7, 16, 35, 70, and 105
- Method of collection of CO2 and volatile organic compounds: The 14CO2 formed and bound to the soda lime was liberated with 18% HCI, trapped in a cocktail of 10 mL ®Carbosorb and 10 mL ®Permafluor and radioassayed. The soda lime contained in the trap attachments was transferred into a 100mL Erlenmeyer flask, 60 mL of 18% HCI was added in droplets via a dropping funnel and the liberated 14CO2 was swept out in a stream of nitrogen for about 30 minutes while agitating. The 14CO2 was absorbed in 3 vials connected in series and filled with 20 mL ice-cooled cocktail each. This was followed by LS-measurement of the samples. Since larger amounts of 14CO2 (i.e. >10%) were formed in the course of study, an identification via [14C]benzoic acid was carried out exemplary. Volatile organic (radioactive) compounds occurring during incubation were trapped by the polyurethane plug located in the trap attachment. The PU-plug was extracted with 15 mL ethylacetate in an ultrasonic bath and the extract was radioassayed.
- Sampling intervals/times for:
> Redox potential/other: On days 0, 3, 7, 16, 35, 70, and 105

Soil No.:

#1

% Recovery:

96.7

Remarks on result:

other: label #1; means of duplicates; after 105 d

Remarks:

for details see "Any other information on results incl. tables" below

Soil No.:

#1

% Recovery:

97.1

Remarks on result:

other: label #2; means of duplicates; after 105 d

Remarks:

for details see "Any other information on results incl. tables" below

Soil No.:

#2

% Recovery:

97.5

Remarks on result:

other: label #1; means of duplicates; after 105 d

Remarks:

for details see "Any other information on results incl. tables" below

Soil No.:

#2

% Recovery:

93

Remarks on result:

other: label #2; means of duplicates; after 105 d

Remarks:

for details see "Any other information on results incl. tables" below

Soil No.:

#1

% Degr.:

11.4

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: value represents portion of unchanged parent that was located in the solids

Soil No.:

#2

% Degr.:

18.1

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: value represents portion of unchanged parent that was located in the solids

Soil No.:

#1

% Degr.:

10.74

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: Sum (means of duplicates) in water phase and soil extracts. Value represents percentage of applied amount of radioactive labeled test substance

Remarks:

label #1; for details see Table 8 under "Any other information on results incl. tables" below and fig. 1 attached

Soil No.:

#2

% Degr.:

16.91

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: Sum (means of duplicates) in water phase and soil extracts. Value represents percentage of applied amount of radioactive labeled test substance

Remarks:

label #1; for details see Table 10 under "Any other information on results incl. tables" below and fig. 2 attached

Soil No.:

#1

% Degr.:

12.02

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: Sum (means of duplicates) in water phase and soil extracts. Value represents percentage of applied amount of radioactive labeled test substance

Remarks:

label #2; for details see Table 9 under "Any other information on results incl. tables" below and fig. 3 attached

Soil No.:

#2

% Degr.:

19.26

Parameter:

radiochem. meas.

Sampling time:

105 d

Remarks on result:

other: Sum (means of duplicates) in water phase and soil extracts. Value represents percentage of applied amount of radioactive labeled test substance

Remarks:

label #2; for details see Table 11 under "Any other information on results incl. tables" below and fig. 4 attached

Soil No.:

#1

DT50:

35 d

Type:

(pseudo-)first order (= half-life)

Temp.:

28 °C

Remarks on result:

other:

Remarks:

According to Timme et al (1986): Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203 (1986). BAYER - Pflanzenschutznachrichten 39/2. 187-203.

Soil No.:

#1

DT50:

10 d

Type:

other: Sqrt 1st order

Temp.:

28 °C

Remarks on result:

other: Best fit

Remarks:

According to Timme et al (1986): Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203 (1986). BAYER - Pflanzenschutznachrichten 39/2. 187-203.

Key result

Soil No.:

#2

DT50:

46 d

Type:

(pseudo-)first order (= half-life)

Temp.:

28 °C

Remarks on result:

other:

Remarks:

According to Timme et al (1986): Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203 (1986). BAYER - Pflanzenschutznachrichten 39/2. 187-203.

Soil No.:

#2

DT50:

18 d

Type:

other: Sqrt 1st order

Temp.:

28 °C

Remarks on result:

other: Best fit

Remarks:

According to Timme et al (1986): Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203 (1986). BAYER - Pflanzenschutznachrichten 39/2. 187-203.

Soil No.:

#1

DT50:

151.6 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Recalculation; Half-life normalized to a temperature of 12 °C following ECHA guidance R.7b (2017)

Soil No.:

#1

DT50:

43.3 d

Type:

other: Sqrt 1st order

Temp.:

12 °C

Remarks on result:

other: Recalculation; Half-life normalized to a temperature of 12 °C following ECHA guidance R.7b (2017)

Key result

Soil No.:

#2

DT50:

199.2 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Recalculation; Half-life normalized to a temperature of 12 °C following ECHA guidance R.7b (2017)

Soil No.:

#2

DT50:

77.9 d

Type:

other: Sqrt 1st order

Temp.:

12 °C

Remarks on result:

other: Recalculation; Half-life normalized to a temperature of 12 °C following ECHA guidance R.7b (2017)

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Pathways for transformation: see attached Fig. 5

Evaporation of parent compound:

no

Volatile metabolites:

yes

Remarks:

CO2

Residues:

yes

Remarks:

The portion of not extracted (i.e. bound) residue in soil increased from values of less than 10% to about 19% (phenyl-UL-14C-label) and to about 32% (cyclohexyl-1-14C-label) at the termination of study.

Details on results:

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

MAJOR TRANSFORMATION PRODUCTS
- CRT: The main metabolite resulting from C-label #1, the 2-chlorophenyltetrazolinone (CRT), was found in the water phase as well as the soil extracts and reached its maximum portion at about 35 to 70 days yielding 39% in alluvial by 32% of applied RA in volcanic ash soil system. Finally, the CRT content declined to about 31% and 26% at day 105.
- CPT: A further metabolite, CPT-Me (M1), reached its maximum at day 105 by about 11% and 13% of applied RA in alluvial and volcanic ash soil system, but was mainly found in the soil extracts.
- CEA: The main metabolite resulting from the cyclohexyl-1-14C-label, cyclohexylethylamine (CEA), was mainly extracted from the soils and reached a maximum portion of 7% and 10% of applied RA at the 16 to 35 days intervals in alluvial and volcanic ash soil system. Finally, the CEA content declined to about 2 and 5% at day 105.

MINOR TRANSFORMATION PRODUCTS
- Further metabolites (i.e. >5%) resulting from both labels were not found.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: At test termination, the 14CO2 values (means of duplicates) were 11.8% (volcanic ash) and 16.0% (alluvial) of applied amount for the phenyl-UL-14C-label. The 14CO2 values (means of duplicates) were 33.3% (volcanic ash) and 43.2% (alluvial) of applied amount for the [cyclohexyl-1-C]-label.

Microbial activity, pH-value, redox potential and oxygen content:

In long-term experiments with micro-ecosystems the measurement of relevant parameters such as biological activity (soil respiration rate), pH-value, redox potential (RP) and oxygen content (DO) is necessary to indicate any change of the living conditions for the microorganisms. The determination of the soil respiration rate in the soils indicated that the systems were biologically active during the entire period of the test. All the measured values ranged from about 19 - 69 mg CO2/h per kg soil (DS). A clear time-dependent tendency was not found. However, the addition of parent compound might influence the height of the soil respiration rate in a positive manner. The pH-value (1 N KCI) measured in the soils of the Bio vessels was more or less constant during the entire study period. The direct records of pH in the soils of the incubated test systems indicated a slight increase from about pH 6 to 7 in both submerged soils. Same tendency was observed by evaluating the pH records of the water phase. Usually, the redox potential (RP) in the supernatant water of both test systems remained at values greater than +40 mV. However, greater variations between different vessels were observed. In general, the minimum of RP (as well as of DO; see later) was measured at interval day 3. Obviously, the problem of a comparatively low oxygen content in the incubator could be resolved quickly and the values increased in the following period of study, again. The measurement of RP in the soils (in the middle of the soil layers) was rather difficult because of the very thin soil layers/vessel. In both the submerged soils the measured RP decreased from positive values to negative values within a few days. This may be caused by sedimentation of the soils and a subsequent lack of oxygen in the unmoved soil layer. In general, oxygen was available in the individual vessels at each sampling time: from 5% to 98% in alluvial system and from 15% to 100% in the volcanic ash system. Thus, the data showed that the water phases were aerobic during the entire incubation period. However, the comparatively low RP and DO values measured at interval day 3 were the reason for opening the incubator slightly. From then on, the oxygen exchange was much better and the values recovered rapidly.

Table 1: Measurement of dissolved oxygen (DO), redox potential (RP) and pH in alluvial soil system

Sampling time [d]	Sample ID	Water phase			Soil
		DO [%]	RP [mV]	pH	RP [mV]	pH
0	Allu/0 days/A	34	168	6.6	34	6.5
	Allu/0 days/B	32	168	6.3	44	6.0
	Allu/0 days/C	38	164	6.4	31	5.9
	Allu/0 days/D	30	171	6.6	32	6.4
3	Allu/3 days/A	11	-30	6.9	-87	6.8
	Allu/3 days/B	21	-50	7.3	-96	7.0
	Allu/3 days/C	11	-32	7.3	-92	7.1
	Allu/3 days/D	5	-54	7.1	-119	7.1
7	Allu/7 days/A	55	104	7.0	-60	6.6
	Allu/7 days/B	67	47	7.8	-85	7.1
	Allu/7 days/C	59	47	7.8	-94	7.2
	Allu/7 days/D	60	62	7.3	-170	6.8
16	Allu/1 6 days/A	72	55	7.0	-138	6.9
	Allu/16 days/B	36	50	7.4	-140	6.9
	Allu/1 6 days/C	83	40	7.4	-113	6.8
	Allu/1 6 days/D	45	57	6.6	-145	6.5
35	Allu/35 days/A	80	11	7.5	-110	6.9
	Allu/35 days/B	81	42	7.9	-120	7.3
	Allu/35 days/C	79	44	7.4	-105	7.1
	Allu/35 days/D	78	-13	7.9	-102	7.0
70	Allu/70 days/A	92	123	7.0	-80	6.8
	Allu/70 days/B	88	87	7.2	-32	7.1
	Allu/70 days/C	87	157	7.5	-54	7.1
	Allu/70 days/D	70	90	6.5	-67	6.6
105	Allu/1 05 days/A	98	126	7.1	-41	7.1
	Allu/1 05 days/B	67	58	6.8	-92	6.5
	Allu/1 05 days/C	77	93	7.3	-59	6.9
	Allu/1 05 days/D	52	85	7.0	-113	7.0

 

Table 2: Measurement of dissolved oxygen (DO), redox potential (RP) and pH in volcanic ash soil system

Sampling time [d]	Sample ID	Water phase			Soil
		DO [%]	RP [mV]	pH	RP [mV]	pH
0	Volc/0 days/A	43	180	6.4	94	5.7
	Volc/0 days/B	35	194	6.9	85	6.1
	Volc/0 days/C	33	175	6.7	98	6.2
	Volc/0 days/D	38	175	6.5	89	6.0
3	Volc/3 days/A	29	14	6.3	-55	6.3
	Volc/3 days/B	19	19	6.1	-36	6.6
	Volc/3 days/C	15	28	6.2	-22	6.6
	Volc/3 days/D	25	28	6.3	-24	6.3
7	Volc/7 days/A	66	64	6.6	-40	6.4
	Volc/7 days/B	64	39	7.2	-66	6.8
	Volc/7 days/C	29	88	6.8	3	7.0
	Volc/7 days/D	42	59	6.5	-62	6.4
16	Volc/1 6 days/A	73	63	6.8	-114	6.5
	Volc/16 days/B	22	56	6.9	-134	6.3
	Volc/1 6 days/C	82	69	6.8	-64	6.4
	Volc/1 6 days/D	47	63	7.0	-78	6.8
35	Volc/35 days/A	83	117	7.2	-54	6.9
	Volc/35 days/B	78	71	7.1	-83	6.8
	Volc/35 days/C	79	11	7.2	-65	6.6
	Volc/35 days/D	80	-31	6.6	-80	6.7
70	Volc/70 days/A	83	145	7.6	-40	6.9
	Volc/70 days/B	87	44	7.2	-65	6.9
	Volc/70 days/C	72	132	7.6	-52	7.3
	Volc/70 days/D	87	84	7.2	-63	7.2
105	Volc/1 05 days/A	93	160	7.3	-20	7.2
	Volc/1 05 days/B	75	12	6.5	-39	6.1
	Volc/1 05 days/C	86	144	7.4	-36	7.1
	Volc/1 05 days/D	100	150	7.6	-12	7.1

Notes on Table 1 & 2: The values of DO and RP during the first three days of study were found to be lower than expected. It was learned that the incubator was closed to much, obviously resulting in an insufficient air/water exchange. Further, the air/water exchange was improved by slightly opening the door of the incubator

Table 3: Soil respiration rate and pH of paddy soils used in Bio vessels [Samples were incubated on a rotary shaker at ca. 200 rpm and at 22 ± 2 °C in the dark. Portions of glucose added: 5,000 mg/kg dry wt for alluvial soil and 7,000 mg/kg dry wt for volcanic ash soil]

Sampling time [d] / ID	Alluvial soil			Volcanic ash soil
	Soil respiration rate [mg CO2/h per kg soil (DS)]		pH	Soil respiration rate [mg CO2/h per kg soil (DS)]		pH
	plus a.i.	without a.i.	(1 N KCI)	plus a.i.	without a.i.	(1 N KCI)
0 d/Bio+/1	49		5.7	27		5.6
0 d/Bio+/2	49		5.7	29		5.8
0 d/Bio-/1		19	5.5		37	5.4
0 d/Bio-/2		20	5.4		38	5.4
105d/Bio+/1	27		5.4	69		5.4
105d/Bio+/2	22		5.3	43		5.4
105d/Bio-/1		19	5.7		33	5.4
105d/Bio-/2		20	5.5		35	5.4

 

Material balances:

The material balances of applied radioactivity (RA) ranged from 93.3% to 98.5% (alluvial soil, label #1), from 91.2% to 97.4% (alluvial soil, label #2), from 96.2% to 100.5% (volcanic ash soil, label #1) and from 91.3% to 99.1% (volcanic ash soil, label #2). A time-dependent tendency was not found. The mean for all the vessels amounted to 96.5% (RSDN = 2.6%). The complete material balances found at all sampling intervals demonstrate that no RA dissipated from the systems during the entire testing period.

Table 4: Material balance for paddy alluvial soil: [phenyl-UL-14C]-test substance

Compartment	Content of radioactivity [% of applied amount] [means of duplicate]
	0 d	3 d	7 d	16 d	35 d	70 d	105 d
Organic volatiles	n.d.	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1
14CO2	n.d.	< 0.1	0.1	1.9	5.9	10.7	16.0
Sum of volatile compounds	n.d.	< 0.1	0.1	1.9	5.9	10.7	16.0
Water phase*	4.1	6.6	12.7	24.0	28.2	30.0	26.8
Soil extract	85.2	84.4	77.4	60.5	47.3	40.6	35.5
Filter paper	0.2	0.1	0.1	< 0.1	0.1	< 0.1	< 0.1
Soil combustion after extraction	8.8	6.1	8.2	10.2	11.8	15.8	18.4
Sum of not extracted residues	9.0	6.2	8.3	10.2	11.9	15.8	18.4
Balance	98.3	97.2	98.5	96.6	93.3	97.1	96.7

* content of water phase (including dissolved carbonates); n.d.: not determined

 

Table 5: Material balance for paddy alluvial soil: [cyclohexyl-1- C]-test substance

Compartment	Content of radioactivity [% of applied amount] [means of duplicate]
	0 d	3 d	7 d	16 d	35 d	70 d	105 d
Organic volatiles	n.d.	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1
14CO2	n.d.	< 0.1	0.5	4.0	11.1	32.8	43.2
Sum of volatile compounds	n.d.	< 0.1	0.5	4.0	11.1	32.8	43.2
Water phase*	3.4	2.0	3.3	4.8	3.6	1.3	0.8
Soil extract	86.9	84.5	76.4	56.8	42.5	25.3	18.4
Filter paper	0.2	0.1	0.1	0.1	0.1	0.1	0.1
Soil combustion after extraction	6.6	10.8	16.8	28.7	33.9	35.0	34.6
Sum of not extracted residues	6.8	10.9	16.9	28.8	34.0	35.1	34.7
Balance	97.1	97.4	97.1	94.4	91.2	94.5	97.1

* content of water phase (including dissolved carbonates); n.d.: not determined

Table 6: Material balance for paddy volcanic ash soil: [phenyl-UL-14C]-test substance

Compartment	Content of radioactivity [% of applied amount] [means of duplicate]
	0 d	3 d	7 d	16 d	35 d	70 d	105 d
Organic volatiles	n.d.	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1
14CO2	n.d.	< 0.1	< 0.1	1.2	4.5	6.5	11.8
Sum of volatile compounds	n.d.	< 0.1	0.1	1.2	4.5	6.5	11.8
Water phase*	3.3	3.5	5.6	12.3	14.9	12.2	12.3
Soil extract	84.3	87.4	83.8	71.4	61.4	61.0	52.7
Filter paper	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Soil combustion after extraction	10.7	8.0	10.9	12.6	15.3	18.1	20.6
Sum of not extracted residues	10.8	8.1	11.0	12.7	15.4	18.2	20.7
Balance	98.4	99.0	100.5	97.6	96.2	97.9	97.5

* content of water phase (including dissolved carbonates); n.d.: not determined

Table 7: Material balance for paddy volcanic ash soil: [cyclohexyl-1- C]-test substance

Compartment	Content of radioactivity [% of applied amount] [means of duplicate]
	0 d	3 d	7 d	16 d	35 d	70 d	105 d
Organic volatiles	n.d.	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1
14CO2	n.d.	0.1	0.4	4.6	14.0	21.9	33.3
Sum of volatile compounds	n.d.	0.1	0.4	4.6	14.0	21.9	33.3
Water phase*	3.4	1.3	1.2	1.7	1.2	1.0	0.6
Soil extract	83.2	87.5	82.0	68.3	50.4	39.5	29.8
Filter paper	0.2	0.1	0.1	0.1	0.1	0.1	0.1
Soil combustion after extraction	12.3	10.1	14.7	21.1	25.6	29.8	29.2
Sum of not extracted residues	12.5	10.2	14.8	21.2	25.7	29.9	29.3
Balance	99.1	99.1	98.4	95.8	91.3	92.3	93.0

* content of water phase (including dissolved carbonates); n.d.: not determined

Distribution of radioactivity:

I) Mineralization (14CO2) and volatile organic compounds:

During the entire study period a significant formation of 14CO2 could be observed in both soil systems and with both 14C-labels used. In general, the portion of 14CO2 trapped was much higher when using the cyclohexyl-1-14C-label. At termination of the test, the 14CO2 recoveries (means of duplicates) were 11.8% (volcanic ash) and 16.0% (alluvial) of applied amount for the phenyl-UL-14C-label, but 33.3% (volcanic ash) and 43.2% (alluvial) of applied amount for the other 14C-label. The identity of 14CO2 was confirmed by means of the Grignard reaction via [14C]benzoic acid . From the before-mentioned data it can be concluded that the test substance is thoroughly degraded and mineralized in the submerged soils. No further volatile (i.e.) organic compounds (each less than 0.04% of applied RA) could be observed in the traps during the entire incubation period.

II) Active ingredient and metabolites in the water layer:

Even quite after starting the test (day 0), the RA content in the overlying water phase was quite low (3.3 - 4.1% of dose) for both soil systems and both labels used. During the entire study period this portion remained low in the case of cyclohexyl-1-14C-label. In the water layer of volcanic ash system a decrease to about 0.6% at interval 105 days was measured, whereas in the water layer of alluvial soil an increase to a maximum portion of 5% at interval day 16 was observed, first. But later, a comparable decrease to a portion of less than 0.8% after 105 days occurred. However in case of the phenyl-UL-14C-label, the portion in the water layer increased to about 15% (volcanic ash) and about 30% (alluvial) until 35 to 70 days. Then a slowly decline of the portion of RA contained in the water phase was measured, already. From the data it can be concluded that the test item was quickly translocated from the water layer into the soil. The DT50 values of the test substance for the supernatant water were less than one hour, that is less than the period from application to day-0-processing. For both 14C-labels a portion of less than 1.4% of applied could be detected in the separated water phase after 3 days. Not any test substance was recovered in the supernatant water after 105 days. Furthermore, the data showed that the main degradation product resulting from a hydrolytic cleavage of the molecule, that is 2-chloro-phenyltetrazolinone (CPT) may be relevant for the overlying waters. The identity of substance was verified by overlapping co-chromatography of water phase with the authentical standard as well as by isolating the respective product from soil extracts and investigating that preparative by LC-MS and LC-MS/MS (ESI/neg). The degradation product CPT reached a maximum portion of 13.0% of applied RA at day 35 in the water phase of volcanic ash soil. Then the portion decreased to 10.2% until the 105 days interval. In the water phase of alluvial soil the CPT reached a maximum portion of 27.5% of applied RA at day 70 and the portion decreased to 23.5% until the 105 days interval. The significant difference of CPT content in the water may be connected to the slowlier

degradation of the test item in the volcanic ash soil. Its higher organic carbon content should lead to a better adsorption of the test item and result in a lower bio-availability.

Further metabolites were not relevant in the water layers, neither resulting from 14C-label #1 nor from 14C-label #2. The 2nd half of the hydrolytic cleavage of the test substance, the cyclohexylethylamine (CEA) was detectable there in quite low portions. All the different individual peak zones corresponded to less than 2% of applied RA.

III) Active ingredient and metabolites in the submerged soils:

Despite having dosed the test substance into the water the RA portion found in the submerged soils processed at day 0 was rather high (93.7 - 95.7% of dose) for both soil systems and 14C-labels used.

During the entire study period the RA in soil continually decreased in case of both 14C-labels and submerged soils used. In the alluvial soil a decrease to 53.9% (#1) and 53.1% (#2) until interval 105 days was measured, whereas a decrease to 73.4% (#1) and 59.1% (#2) until interval 105 days was observed in the volcanic ash soil, only. More significant was the decrease of extractable RA during the incubation period. In the alluvial soil that portion decreased from 85.2 to 35.5% (#1) and from 86.9 to 18.4% (#2) until interval 105 days, whereas that portion decreased from max. 87.4 (day 3) to 52.7% (#1) and from 87.5 (day 3) to 29.8% (#2) until interval 105 days in the volcanic ash soil. Generally, the portion of extractable RA from the soils after 105 days was much lower in case of cyclohexyl-1-14C-label (#2). Thus, higher portions of 14CO2 as well as of bound residues, but lower residues in the water phase occurred correspondingly in case of 14C-label #2. During the entire study period the RA in soil continually decreased in case of both 14C-labels and submerged soils used. In the alluvial soil a decrease to 53.9% (#1) and 53.1% (#2) until interval 105 days was measured, whereas a decrease to 73.4% (#1) and 59.1% (#2) until interval 105 days was observed in the volcanic ash soil, only. More significant was the decrease of extractable RA during the incubation period. In the alluvial soil that portion decreased from 85.2 to 35.5% (#1) and from 86.9 to 18.4% (#2) until interval 105 days, whereas that portion decreased from max. 87.4 (day 3) to 52.7% (#1) and from 87.5 (day 3) to 29.8% (#2) until interval 105 days in the volcanic ash soil. Generally, the portion of extractable RA from the soils after 105 days was much lower in case of cyclohexyl-1-14C-label (#2). Thus, higher portions of 14CO2 as well as of bound residues, but lower residues in the water phase occurred correspondingly in case of 14C-label #2. From the data it can be concluded that the test substance is quickly adsorbed and thoroughly degraded and mineralized in the submerged soils. There is no potential for persistence or accumulation in the paddy soils. In the submerged alluvial soil the residues of the test substance (expressed as % of dosed RA) continually decreased from 79.8 to 10.7% (#1) and from 84.3 to 12.0% (#2) until interval 105 days. In the volcanic ash soil this portion decreased from max. 76.4 (day 3) to 16.9% (#1) and from 80.4 to 19.3% (#2) until interval 105 days. In the paddy soils used no larger difference was found in the potential for degrading the test item. The DT50 values of the test item in the submerged soils are closely related to those of the total test system. The identity of parent compound was verified by overlapping co-chromatography of soil extracts with the authentical standard as well as by isolating the respective product from soil extracts and investigating that preparative by LC-MS (ESI/pos) and 1HNMR. Furthermore, the analyses showed that both the main degradation products resulting from hydrolytic cleavage of the molecule, 2-chlorophenyltetrazolinone (CRT) as well as cyclohexylethylamine (CEA) may be relevant residues in the submerged soils for short term. The identity of CEA was verified by overlapping co-chromatography of soil extracts with the authentical standard as well as by isolating the respective product from soil extracts and investigating that preparative by LC-MS and LC-MS/MS (ESI/pos). The degradation product CPT reached a maximum portion of 13.0% of applied RA at day 35 in the extracts of alluvial soil. Until the 105 days interval

the portion decreased to 8.0%, already. In the volcanic ash soil the CPT reached a maximum portion of 19.0% of applied RA at day 35 and the portion decreased to 15.4% until 105 days. The cyclohexylethylamine (CEA) reached a maximum portion of 6.3% of applied RA at day 35 in the extracts of alluvial soil. Until the 105 days interval the portion decreased to 2.3%. In the volcanic ash soil the CEA reached a maximum portion of 9.2% of applied RA at day 16 and the portion decreased to 5.3% until the 105 days interval. Obviously, this part of the parent compound molecule is more intensively degraded in soil as the CPT-part. Apparently from that period on when the CRT content exceeded its maximum in the submerged soils, that is after 35 days of incubation, a further metabolite

resulting from 14C-label #1 was detected in the soil extracts in relevant amounts. In the subsequent study period and concurrent with the decrease of CPT, the portions of M1 increased to 8.9% in alluvial soil and 12.2% in the volcanic ash soil until 105 days. Therefore, it was concluded that the metabolite M1, that was less polar than CPT (consequently less important for the water layers), may have been formed from CPT. In fact, the structure of M1 was verified as CPT-Me. The identity of M1 was verified by overlapping co-chromatography of soil extracts with the authentical standard as well as by isolating the respective product from soil extracts and investigating preparatives by LC-MS and LC-MS/MS (ESI/ pos). Further metabolites were not relevant in the soil extracts, neither resulting from 14C-label #1 nor from 14C-label #2. Each of the various individual peak zones including start RA on TLC corresponded to less than 5% of RA dosed to the systems.

IV) Bound residue:

For both soil systems and labels the portion of bound residue in soil increased from values of less than 10% of applied RA to about 19% (phenyl-UL-14C-label) and to about 32% (cyclohexyl-1-14C-label) at study termination. The significant difference found for both 14C-labels indicates that the bound residue is formed mainly by cleavage products and not by slightly changed metabolites. Always, the content of RA in the paper filters was very low: each less than 0.3% of applied dose. This portion is regarded as bound residue, too. The higher amounts of bound residue formed in case of the cyclohexyl-1-14C-label were further characterized by means of a more stringent extraction. About 7 -10% of dosed RA (corresponding to about 1/3 of respective bound residue) could be extracted further from the soil samples used. In those extracts a small portion was detected as parent compound, whereas the main portion corresponded to CEA. Both substances were regarded as relevant residues in the submerged soils, already.

V) Active ingredient and metabolites in the paddy soil systems:

For the degradation of the test substance and the formation/degradation of metabolites in the different soils and for the different labels see figures 1 to 4 atatched below.

VI) Half-lives:

a) Half-lives of test substance in the water layers:

It can be concluded that the test substance is very quickly eliminated from the water body and translocated into the soil. The DT50 values for the surface water were less than the period until first processing, that is less than 1 hour.

b) Half-lives of test substance in the submerged soils:

It can be concluded that the disappearance time of the test substance in the submerged soils is quite the same as that calculated for the total system. This is justified, because the predominant portion of residues were located in the submerged soils, always.

c) Half-lives of the test substance in the paddy soil system:

The DT50-values for the test substance in the paddy soil system were estimated by the method of Timme et al. [Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203 (1986)]. In Table 7 below the various results are summarized:

Table 8: Summary of DT50 calculations for the test substance

Paddy soil system	14C-label	Half-life [days]	Function	Remark
Alluvial	Phenyl-UL	34.5	1st order
	Cyclohexyl-1-	36.1	1st order
	Phenyl-UL	10.0	Sqrt 1st order	Best fit
	Cyclohexyl-1-	10.7	Sqrt 1st order	Best fit
Volcanic ash	Phenyl-UL	44.7	1st order
	Cyclohexyl-1-	48.0	1st order
	Phenyl-UL	16.8	Sqrt 1st order	Best fit
	Cyclohexyl-1-	18.7	Sqrt 1st order	Best fit

 

Table 9: Content of test substance and relevant metabolites in paddy alluvial soil (soil #1). Sum (means of duplicates) in water phase and soil extracts [n.d. = not detected]

Sampling Interval [d]	[Phenyl-UL-14C]-test substance [% of applied amount]
	Test substance	CPT	M1	Start	Others
0	82.53	0.51	n.d.	1.28	4.97
3	74.55	8.09	n.d.	2.02	6.45
7	66.94	16.10	n.d.	1.90	4.97
16	42.99	30.27	n.d.	4.08	6.75
35	27.37	38.28	2.58	1.99	5.05
70	18.16	39.11	6.01	3.04	4.19
105	10.74	31.47	10.64	3.98	5.49

 

Table 10: Content of test substance and relevant metabolites in paddy alluvial soil (soil #1). Sum (means of duplicates) in water phase and soil extracts [n.d. = not detected]

Sampling Interval [d]	[Cyclohexyl-1-14C]-test substance [% of applied amount]
	Test substance	CEA	Start	Others
0	87.36	0.06	n.d.	2.95
3	78.89	2.87	0.19	4.94
7	61.59	4.97	0.38	12.69
16	39.92	6.77	0.60	13.76
35	28.11	7.00	0.45	9.74
70	18.58	2.96	0.07	4.67
105	12.02	2.34	0.19	4.40

 

Table 11: Content of test substance and relevant metabolites in paddy volcanic ash soil (soil #2). Sum (means of duplicates) in water phase and soil extracts [n.d. = not detected]

Sampling Interval [d]	[Phenyl-UL-14C]-test substance [% of applied amount]
	Test substance	CPT	M1	Start	Others
0	78.50	1.73	n.d.	1.74	5.61
3	78.91	7.40	n.d.	4.79	0.47
7	70.98	12.03	n.d.	1.22	5.26
16	51.28	27.08	n.d.	4.15	1.09
35	33.51	32.01	5.67	1.87	3.18
70	27.32	28.52	11.09	4.73	1.39
105	16.91	25.64	13.06	3.16	6.31

 

Table 12: Content of test substance and relevant metabolites in paddy volcanic ash soil (soil #2). Sum (means of duplicates) in water phase and soil extracts [n.d. = not detected]

Sampling Interval [d]	[Cyclohexyl-1-14C]-test substance [% of applied amount]
	Test substance	CEA	Start	Others
0	83.69	0.08	0.00	2.92
3	78.35	3.97	0.29	6.38
7	63.07	6.78	0.47	12.82
16	48.46	9.47	0.50	11.48
35	35.43	8.39	0.48	7.03
70	26.92	8.25	0.04	5.04
105	19.26	5.40	0.30	5.33

 

Description of key information

Half-life in soil: 46 days at 28 °C (Guideline for the Experiment of Pesticide Residue in Soil, Environmental Agency of Japan (1978) and B59 NOSAN No. 4200, MAFF Japan (1985)

Half-life in soil: 199 days at 12 °C (re-calculated based on ECHA (2017) Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7b: Endpoint specific guidance. Version 4.0

Key value for chemical safety assessment

Half-life in soil:

199 d

at the temperature of:

12 °C

Additional information

The key study available (1997) describes the degradation and partitioning behavior of [phenyl-UL-14C] and [cyclohexyl-1-14C]-labeled test substance and its metabolites occurring in submerged soil (alluvial and volcanic ash soil) and water layer under paddy conditions without plants. Basis for performing this study was the guideline of the Environmental Agency of Japan “Guideline for the Experiment of Pesticide Residue in Soil” of the year 1978 and the “B59 NOSAN No. 4200” of Japan MAFF of the year 1985.

After dosing 0.28 kg a.i./ha the test vessels were incubated at a temperature of 28 °C under darkness over a maximum period of 105 days. At each interval (on days 0, 3, 7, 16, 35, 70, and 105), the supernatant water and soil were separated and analyzed by means of thin-layer chromatography; the content of radioactivity (RA) was determined by liquid scintillation measurement. A metabolic pathway is proposed by evaluating all the results.

The material balance established for each individual vessel and sampling interval ranged from 91.2% to 100.5% of applied RA (means of duplicates). The mean for all the vessels was 96.5% (RSDN = 2.6%). The complete material balance demonstrates no significant losses from the systems occurring during the test period.

During the entire study period a significant formation of 14CO2 could be observed in both soil systems and with both 14C-labels used. At test termination, the 14CO2 values (means of duplicates) were 11.8% (volcanic ash) and 16.0% (alluvial) of applied amount for the phenyl-UL-14C-label, but 33.3% (volcanic ash) and 43.2% (alluvial) of applied amount for the [cyclohexyl-1-C]-label. No further volatile radioactive products occurred in the traps during the entire study period.

Even quite after starting the test (day 0), the RA content in the overlying water phase was quite low (3.3 - 4.1% of dose) for both soil systems and both labels used. During the entire study period this portion remained very low in the case of the cyclohexyl-1 -14C-label (<1% after 105 days). However, in case of the other label that portion increased to about 15% (volcanic ash) and about 30% (alluvial) until 35 to 70 days. Afterwards, a decline of the portion was measured.

Generally, the portion of not-extracted (that is bound) residue in soil increased from values of less than 10% to about 19% (phenyl-UL-14C-label) and to about 32% (cyclohexyl-1-14C-label) at the termination of study.

After 105 days of incubation the portion of unchanged parent that was located in the solids, amounted to 11.4% (alluvial) and 18.1% (Volcanic ash) of applied dose, only. The main metabolite resulting from C-label #1, the 2-chlorophenyltetrazolinone (CRT), was found in the water phase as well as the soil extracts and reached its maximum portion at about 35 to 70 days yielding 39% in alluvial by 32% of applied RA in volcanic ash soil system. Finally, the CRT content declined to about 31% and 26% at day 105. A further metabolite, CPT-Me (M1), reached its maximum at day 105 by about 11% and 13% of applied RA in alluvial and volcanic ash soil system, but was mainly found in the soil extracts. Further metabolites (i.e. >5%) resulting from the before-mentioned label were not found. The main metabolite resulting from the cyclohexyl-1-14C-label, cyclohexylethylamine (CEA), was mainly extracted from the soils and reached a maximum portion of 7% and 10% of applied RA at the 16 to 35 days intervals in alluvial and volcanic ash soil system. Finally, the CEA content declined to about 2 and 5% at day 105. Further metabolites (i.e. >5%) resulting from the 2nd label were not found.

DT50 values for the test substance in the paddy soil systems (submerged soil plus water) were estimated and are given in the table below. The test substance was found to be well degradable in both paddy soil systems tested. The DT50 values for the surface water were found to be less than 1 hour, that is less than the period from application to day-0-processing.

Table: Summary of DT50 calculations for the test substance based on the test temperature of 28 °C.

Paddy soil system	14C-label	Half-life [days]	Half-life [days] (mean)	Function	Remark*
Alluvial	Phenyl-UL	34.5	35.0	1st order
	Cyclohexyl-1-	36.1		1st order
	Phenyl-UL	10.0	10.0	Sqrt 1st order	Best fit
	Cyclohexyl-1-	10.7		Sqrt 1st order	Best fit
Volcanic ash	Phenyl-UL	44.7	46	1st order
	Cyclohexyl-1-	48.0		1st order
	Phenyl-UL	16.8	18	Sqrt 1st order	Best fit
	Cyclohexyl-1-	18.7		Sqrt 1st order	Best fit

* according to Timme et al (1986, vers. 2): Statistical Interpretation and Graphic Representation of the Degradational Behaviour of Pesticide Residues II. Bayer - Pflanzenschutznachrichten 39/2. 187-203.

For short term, the main degradation product resulting from a hydrolytic cleavage, the 2-chlorophenyltetrazolinone (CRT), may be relevant for the overlying waters. However, residues of a transformation product of CPT, that is CPT-Me, as well as residues from the other part of the parent compound molecule, that is cyclohexylethylamine (CEA), are expected to occur in the submerged soils, mainly. Additionally, the data showed that the CEA-part of parent compound molecule is more rapidly and thoroughly degraded than the CPT-part. This observation is matched by a higher portion of bound residue in soil due to an intensive metabolization and a subsequent incorporation of 14C-products into the organic matter of soil.

In the study report it was concluded that the test substance is quickly eliminated from the water phase and thoroughly degraded and mineralized in the submerged soils at a test temperature of 28 °C. Under these conditions there is no potential for persistence or accumulation in the paddy soil environment: the 1st order half-life in both tested paddy soils amounted to 5 to 6 weeks, the best fit half-life (sqrt. 1st order) amounted to 2 to 3 weeks.

However, according to the ECHA document "Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7b: Endpoint specific guidance. Version 4.0" (2017) the preferred temperature for simulation degradation studies is 12 °C, which corresponds to the average temperature of European surface waters. Where this condition is not met a temperature correction should be considered based on the Arrhenius equation and half-lives be re-calculated. For first order kinetics this was done and half-lives of 35 and 46 days (at 28 °C) were re-calculated to 152 and 199 days (at 12 °C), respectively.

A second soil simulation study is available (1995). This study was conducted as a preliminary test and did not follow any guideline or GLP principles. However, this study is well-documented and thus reported here.

The metabolism of the test substance in paddy soils was studied with radiolabeled compounds. Same soils and labels as in the key study were used and soils were treated with 0.3 mg/kg of radiolabeled test substance. The soils were incubated in the dark at 28 °C under submerged condition. The test substance was metabolized by microorganisms in paddy soil. Half-life of label #1 test substance was 28 days. 2.8% of carbon dioxide was evolved at 56 day. Dissipation of parent compound in Ushiku soil was faster than that in Ryugasaki soil. The half-life was 15 days. 0.8% of carbon dioxide was evolved at 56 day. Bound residue increased gradually up to 7.7% - 8.3% at 56 day in both soils. The metabolism of label #2 test substance was as fast as that of label #1 (half-life of 35 days). 13.2% of carbon dioxide was evolved at 56 day. Bound residue amounted to 33.6% at 56 day. Main metabolite in paddy soil was CPT which was detected as 50% at 56 day. In the experiment with label #2, 13.5% of CEA was detected in the peak of concentration. In the label #1 experiment, approximate 20% of unknown metabolite was also detected in the peak of concentration. Effect of oxygen supply on the metabolism of CPT in the soil was studied. The metabolism was enhanced when the test vessels were shaken to help oxygen supply. The test substance was considered to be metabolized by way of urea cleavage to form CPT and CEA, which were further metabolized oxidatively to carbon dioxide. These results are in congruence with the key study. Half-lives established at 28 °C are of the same magnitude as in the key study. No re-calculation to 12 °C was done as values from the key study represent worst case.