Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2006 to 14 Sep 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

17 July 1992

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of activated sludge: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater.
- Pretreatment: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. During holding, the sludge was aerated at room temperature until use.
- Concentration of sludge: A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

102 mg/L

Based on:

test mat.

Remarks:

Average concentration of two replicates

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 22 ˚C, maintained with a built-in thermostat and checked once per week.
- pH: 7.3 - 7.5 (Starts); 7.3 - 7.7 (end)
- pH adjusted: The pH was adjusted from 8.0 to 7.4 with a diluted hydrochloric acid solution.
- Test water preparation and composition: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the followirlg stock solutions:
a) KH2PO4 (8.50 g/L), K2HPO4 (21.75 g/L), Na2HPO4 x 2H2O (33.40 g/L), NH4CI (0.50 g/L). The pH of this solution was 7.4.
b) MgSO4 x 7H2O (22.50 g/L)
c) CaCl2 x 2H2O (36.40 g/L)
d) FeCb x 6H2O (0.25 g/L, stabilized with one drop of concentrated HCI per liter)
To obtain the final test water, 10 mL of stock solution a) and 1 mL each of stock solutions b) - d) were combined and made up to 1000 mL with purified water.
- Suspended solids concentration: 30 mg dry material/L
- Continuous darkness: Yes
- Electro-chemical analysis process: The biodegradation process consumes the dissolved oxygen in the liquid and generates CO2. The CO2 is adsorbed by soda lime and the total pressure decreases in the airtight test 'flasks. 'The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.

TEST SYSTEM
An overview of the test concentrations is provided in Table 1 in ‘Any other information on materials and methods incl. tables'.
- Culturing apparatus: 500-mL flasks (incubated under continuous stirring)
- Number of culture flasks/concentration: 2
- Number of culture flasks/negative control: 2
- Number of culture flasks/procedure control: 2
- Number of culture flasks/abiotic control: 1
- Number of culture flasks/positive control: 1
- Final volume per culture flask: 250 mL

SAMPLING
- Oxygen consumption was recorded manually by taking a daily reading at least on each working day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Aerobic activated sludge was used as the inoculum. There is no test substance or reference item in the system.
- Procedure control,: the reference item, sodium benzoate, (100 mg/L) was tested.
- Abiotic sterile control: The abiotic control contained test material (100 mg/L) and mercury dichloride (10 mg/L), without activated sludge..
- Toxicity control: The toxicity control contained both test material (99 mg/L) and the reference item sodium benzoate (100 mg/L).

Reference substance:

benzoic acid, sodium salt

Remarks:

100 mg/L (corresponding to a theoretical oxygen demand of 167 mg O2/L)

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

An overview of the results is provided in Table 2 and Table 3 in 'Any other information on results incl. tables'.
BIODEGRADATION OF THE TEST ITEM
The percent biodegradation of the test item was calculated based on the theoretical oxygen demand of 2.00 mg O2/mg test item without nitrification (ThODNH4), and 2.54 mg O2/mg test item with nitrification (ThODNo3). The oxygen consumption of the test item test item in the test media was in the range of the oxygen consumption of the inoculum controls. Consequently, test item was not biodegradable under the test conditions within 28 days. However, in the toxicity control an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was observed, suggesting that the test item was co-metabolized in the presence of sodium benzoate.
- Abiotic control: No degradation of the test item occurred in the abiotic control under the test conditions within 28 days.

BIODEGRADATION IN THE TOXICITY CONTROL
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the ThOD of the test item (ThODNH4 or ThODNO3) and the reference item. Up to about Exposure Day 4, the course of biodegradation in the toxicity control was similar to the two procedure controls, containing only the reference item. Thus, the oxygen consumption durirrg the first four days can primarily be attributed to the biodegradation of the reference item. From Exposure Day 5 to the end of the test, an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was observed in the toxicity control, suggesting that the test item was biodegraded in the presence of sodium benzoate. This process is described in literature as co-metabolism or co-oxidation, and describes the concomitant oxidation of a non-growth substrate during growth of a microorganism on a utilizable carbon and energy source. Within 14 days of exposure, biodegradation amounted to 76% and 66% based on the ThODNH4, and the ThODNO3, respectively. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 99 mg/L because biodegradation in the toxicity control was > 25% within 14 days.

Results with reference substance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand (ThOD) of 1.67 mg O2/mg reference item. In the procedure control, the reference item was degraded by 88% by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by 93%.

Table 2. Oxygen consumption in the test flasks

Time (days)	Cumulative oxygen consumption (mg/L)
	Test item		lnoculum control		Procedure control		Abiotic control	Toxicity control
	Replicate no.		Replicate no.		Replicate no.		Replicate no.	Replicate no.
	1	2	1	2	1*	2	1	1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28	0 0 -- 0 0 0 0 0 2 2 2 2 2 4 4 4 6 6 6 6 6 6 6 6 -- 6 6 6 6	0 0 -- 0 0 0 1 2 2 2 2 2 2 5 5 6 7 7 7 7 7 7 7 7 -- 7 7 7 7	0 0 -- 0 0 0 0 0 0 0 0 0 1 3 3 4 5 5 5 5 5 5 5 5 -- 5 5 5 5	0 0 -- 0 0 0 2 2 2 2 3 4 4 7 7 8 10 10 10 10 10 10 10 10 -- 10 10 11 11	-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --	0 8 -- 111 123 128 134 138 141 144 146 148 148 152 152 154 157 157 157 157 157 159 159 161 -- 161 161 161 163	0 0 -- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -- 0 0 0 0	0 16 -- 112 132 148 166 178 188 202 220 236 250 270 283 299 307 319 326 336 347 356 362 374 -- 390 398 406 413

-- No reading taken

* Replicate no. 1 was not considered in the experimental evaluation, because erratic results were obtained due to a leak in the respective measuring cell. This has no detrimental impact on the study, since only one procedure control is required by the testing guidelines.

 

Table 3. Biodegradation in the test flasks

Time (days)	Percentage Biodegradation1
	Test item based on				Procedure control based on		Toxicity control based on
	ThOD NH4		ThOD NO3		ThOD		ThOD NH4	ThOD NO3
	Replicate no.		Replicate no.		Replicate no.		Replicate no.
	1	2	1	2	1*	2	1
0	0	0	0	0	--	0	0	0
1	0	0	0	0		5	4	4
2	--	--	--	--		--	--	--
3	0	0	0	0		66	31	27
4	0	0	0	0		73	36	32
5	0	0	0	0		76	41	35
6	0	0	0	0		79	45	39
7	0	0	0	0		82	48	42
8	0	0	0	0		84	51	45
9	0	0	0	0		85	55	48
10	0	0	0	0		86	60	52
11	0	0	0	0		87	64	56
12	0	0	0	0		87	68	59
13	0	0	0	0		88	73	63
14	0	0	0	0		88	76	66
15	-1	0	-1	0		88	80	70
16	-1	0	-1	0		89	82	72
17	-1	0	-1	0		89	85	74
18	-1	0	-1	0		89	87	76
19	-1	0	-1	0		89	90	79
20	-1	0	-1	0		89	93	81
21	-1	0	-1	0		91	95	83
22	-1	0	-1	0		91	97	85
23	-1	0	-1	0		92	100	88
24	--	--	--	--		--	--	--
25	-1	0	-1	0		92	105	91
26	-1	0	-1	0		92	107	93
27	-1	0	-1	0		91	109	95
28	-1	0	-1	0		93	111	97
Mean (Day 28)	-1		-1

1 Corrected for the mean oxygen uptake of the inoculum controls

-- Not determined

* Replicate no. 1 was not considered in the experimental evaluation, because erratic results were obtained due to a technical defect. This has no detrimental impact on the study, since only one procedure control is required by the testing guidelines.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In a 28-day ready biodegradability study in accordance with OECD TG 301 F, no biodegradation of the test substance was observed. Thus, it is concluded that the test substance is not biodegradable under the test conditions.

Executive summary:

The ready biodegradability of the test item was investigated using aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fijllinsdorf, Switzerland) treating predominantly domestic wastewater. The test was conducted according to EU Commission Directive 92/69 EEC, C.4-D and OECD TG 301 F and in compliance with GLP criteria. The test sludge was incubated with 102 mg/L of the test substance (2 replicates) at 22 °C in the darkness for 28 days under continuous stirring. An inoculum control (2 replicates), a procedure control (100 mg/L sodium benzoate; 2 replicates), an abiotic control (100 mg/L the test substance and 10 mg/L mercury dichloride; 1 replicate) and a toxicity control (99 mg/L the test substance and 100 mg/L sodium benzoate; 1 replicate) were incubated under the same conditions as the test substance. Oxygen consumption was recorded daily reading at least on each working day. The biodegradation was calculated based on the biochemical oxygen demand of the test or reference item and the theoretical oxygen demand of the test or reference item, without or with nitrification.

In the procedure control, the reference item (sodium benzoate) was degraded by 88% on Day 14 and reached a biodegradation of 93% by the end of the test (Day 28), thus confirming suitability of the activated sludge. In the toxicity control, no inhibitory effect on the biodegradation of the reference item was determined. Thus, the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 99 mg/L. After 28 days incubation, the oxygen consumption of the test item in the test media was in the range of the oxygen consumption of the inoculum controls. Consequently, the test item was not biodegradable under the test conditions within 28 days. However, in the toxicity control, an oxygen consumption exceeding the ThOD of the reference item was observed, suggesting that the test item was co-metabolized in the presence of sodium benzoate. No degradation of the test item occurred in the abiotic control under the test conditions during the test. Based on these findings, it is concluded that the test substance is not biodegradable under the test conditions.

Description of key information

The test substance is concluded to be "not biodegradable" under the test conditions, freshwater, aerobic activated sludge, EU Commission Directive 92/69 EEC, C.4-D and OECD TG 301F, Seyfried 2006

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

There is one ready biodegradability study available, which followed EU Commission Directive 92/69 EEC, C.4-D and OECD TG 301 F and complied with GLP criteria. The ready biodegradability of the test item was investigated using aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fijllinsdorf, Switzerland) treating predominantly domestic wastewater. The test sludge was incubated with 102 mg/L of the test substance (2 replicates) at 22 °C in the darkness for 28 days under continuous stirring. An inoculum control (2 replicates), a procedure control (100 mg/L sodium benzoate; 2 replicates), an abiotic control (100 mg/L the test substance and 10 mg/L mercury dichloride; 1 replicate) and a toxicity control (99 mg/L the test substance and 100 mg/L sodium benzoate; 1 replicate) were incubated under the same conditions as the test substance. Oxygen consumption was recorded daily and read at least on each working day. The biodegradation was calculated based on the biochemical oxygen demand of the test or reference item and the theoretical oxygen demand of the test or reference item, without or with nitrification. After 28 days of incubation, no biodegradation of the test substance was observed. Thus, it is concluded that the test substance is not biodegradable under the test conditions.