Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th April 2011 - 3rd August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant, D-31137 Hildesheim
- Storage length: 2 days
- Pretreatment: Washed twice with autoclaved tap water, filled with mineral water and aerated for 4 hours. The sludge was then homogenised with a blender and the supernatant decanted. It was then aerated for 2 days in CO2 free air
- Initial inoculation: 0.2 mL inoculum per test vessel

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Initial conc.:

2.91 other: mg O2/mg

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 D
- Test temperature: 20-22°C
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD bottles with glass stopper (volume ca. 300 mL, not headspace)
- Measuring equipment: pH-Meter, Oximeter, Incubator

SAMPLING
- Sampling frequency: 7, 14, 21 and 28 day

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: YesTEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 D
- Solubilising agent (type and concentration if used): Not used
- Test temperature: 20-22°C
- pH: 7.27 – 7.29 (test start) 6.75 – 7.21 (test end)
- pH adjusted: No
- Aeration of dilution water: yes
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: BOD bottles with glass stopper (volume ca. 300 mL, no headspace)
- Number of culture flasks: Two replicate samples for test item, functional control, toxicity control and inoculum control
- Measuring equipment: pH-Meter, Oximeter, Incubator
- Test performed in closed vessels due to significant volatility of test substance: Closed bottle test conducted

SAMPLING
- Sampling frequency: 0, 7, 14, 21 and 28 days
- Sampling method: Oxygen concentration of duplicates of the test item, functional control and inoculum control was measured on all sampling days. The toxicity control was measured on days 0, 14 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Results with reference substance:

The reference substance degraded by 70% after 7 days, 69% after 14 days, 79% after 21 days and 68% after 28 days.

Oxygen Determination of the Inoculum Control and Functional Control

Study Day (d)	Inoculum Control				Function Control, 5 mg/L)
					ThOD: 1.67 mg O2/mg
	O2 (mg.L)				O2 (mg/L)			BOD		degr.
	C1	C2	mv	O2 -depl.	R1	R2	mv	(mg O2/L)	(mg O2/mg)	(%)
0	8.55	8.58	8.57	-	8.58	8.59	8.59	-	-	-
7	7.94	7.75	7.85	0.72	1.98	2.08	2.03	5.84	1.17	70
14	7.43	7.50	7.47	1.10	1.50	1.97	1.74	5.75	1.15	69
21	7.33	7.45	7.39	1.18	(7.78)	0.81	0.81	6.60	1.32	79
28	7.8	6.91	7.05	1.52	0.62	2.24	1.43	5.64	1.13	68

mv = mean value; degr. = degradation; depl. = depletion; ThOD = Theoretical oxygen demand; BOD = biological oxygen demand; n.d. = not determined; ( ) = outlier, not included in calculations.

Oxygen Determination of the Test Item and the Toxicity Control

Study day (d)	Test Item: 3 mg/L						Toxicity Control
							1.5 mg/L test item + 2.5 mg/L reference item
	ThOD: 2.91 mg O2/mg						ThOD 2.15 mg O2/mg
	O2 (mg/L)			BOD		degr.	O2 (mg/L)			BOD		degr.
	P1	P2	mv	(mg O2/L)	(mg O2/mg)	(%)	T1	T2	mv	(mg O2/L)	(mg O2/mg)	(%)
0	8.58	8.57	8.58	-	-	-	8.57	8.57	8.57	-	-	-
7	7.66	7.61	7.64	0.22	0.07	2	n.d.	n.d.	-	3.40	0.85	40
14	7.03	7.07	7.05	0.43	0.14	5	4.13	4.00	4.07	-	-	-
21	6.99	7.14	7.07	0.33	0.11	4	n.d.	n.d.	-	3.08	0.77	36
28	6.78	7.02	6.90	0.16	0.05	2	4.24	3.69	3.97	-	-	-

mv = mean value; degr. = degradation; depl. = depletion; ThOD = Theoretical oxygen demand; BOD = biological oxygen demand; n.d. = not determined; ( ) = outlier, not included in calculations.

Biodegradation of the Test Item in Comparison to the Functional Control and the Toxicity Control

	Biodegradation (%)
	Study day (d)
	7	14	21	28
Test item 3 mg/L	2	5	4	2
Functional control 5 mg/L	70	69	79	68
Toxicity Control 1.5 mg/L Test item + 2.5 mg/L Reference Item	n.d.	40	n.d.	36

n.d. = not determined

Validity criteria fulfilled:

yes

Remarks:

Yes, except O2 consumption in inoculum control was 1.52 mg/L after 28 days. This is slightly above the recommended value and is not considered to have impacted on the integrity of the study.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item reached maximum degradation of 5% after 14 days, and was shown to have degraded by 2% at 28 days. The test item is not readily biodegradable.

Executive summary:

The study is GLP compliant and conducted in accordance with OECD guideline 301 D. An inoculum control, functional control and toxicity control were all included. The validity criteria were met, apart from oxygen depletion in the inoculum control was 1.52 mg dissolved oxygen/L after 28 days, slightly above the 1.5 mg O2/L limit. However, this is not considered to have affected the integrity of the study. The study is considered to be reliable without restrictions (Klimisch 1)..

The test item reached maximum degradation of 5% after 14 days, and was shown to have degraded by 2% at 28 days. The test item is not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: German Environmental Agency (1990) (BODIS Test)

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 10708

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant of D-Pforzheim
- Storage length: One week
- Pretreatment: Washed twice by centrifugation (10 min., 6000 rpm) with mineral solution
- Concentration of sludge: 3.0 g/L

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 20°C
- pH: 6.86 – 7.60
- Aeration: 15 minutes prior to the test and after each measurement
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Closed glass bottles
- Number of culture flasks: Two replicates for test item, blank and reference item. One replicate for toxicity control.
- Method used to create aerobic condition: The test vessels were closed glass bottles with 2/3 of aqueous test mixture and 1/3 air. They were shaken continuously to assure steady state oxygen partitioning between liquid and gas phase

- Measuring equipment: Oximeter

SAMPLING
- Sampling frequency: 7, 14, 21 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: 0.5 ml sodium benzoate stock solution and 0.0076 g test item

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

7.5

Sampling time:

28 d

Details on results:

The toxicity control demonstrated toxic effects of the test substance to the microbial population of the test system.

Results with reference substance:

The reference substance sodium benzoate was degraded to 71.4 % of its initial concentration after 28 days.

Degradation Rates after 7 Days

Study Group	Oxygen Content after 0 d	Ur	BOD (mg O2)	% Degradation	Cumul. BOD (mg O2)	Cumul. Degradation (%)
T1	8.90	0.000	0.000	0.0	0.000	0.0
T2	8.90	0.000	0.000	0.0	0.000	0.0
TC	8.85	0.000	0.000	0.0	0.000	0.0
R1	8.30	0.253	7.540	36.1	7.540	36.1
R2	7.95	0.277	8.247	39.5	8.247	39.5

T = test item; TC = toxicity control; R = reference substance; cumul. = cumulative ; U_r = relative uptake of oxygen in the water phase

Degradation Rates after 14 Days

Study Group	Oxygen Content after 0 d	Ur	BOD (mg O2)	% Degradation	Cumul. BOD (mg O2)	Cumul. Degradation (%)
T1	8.45	0.021	0.617	2.9	0.6170.000	2.9
T2	8.30	0.026	0.784	3.9	0.784	3.9
TC	8.55	0.020	0.610	1.5	0.610	1.4
R1	8.75	0.254	7.578	36.3	15.117	72.4
R2	8.35	0.159	4.729	22.7	12.975	62.2

T = test item; TC = toxicity control; R = reference substance; cumul. = cumulative ; U_r= relative uptake of oxygen in the water phase

Degradation Rates after 21 Days

Study Group	Oxygen Content after 0 d	Ur	BOD (mg O2)	% Degradation	Cumul. BOD (mg O2)	Cumul. Degradation (%)
T1	8.85	0.023	0.673	3.2	1.291	6.1
T2	8.85	0.000	0.000	0.0	0.784	3.9
TC	8.55	0.000	0.000	0.0	0.610	1.5
R1	8.85	0.000	0.000	0.0	15.117	72.4
R2	8.85	0.000	0.000	0.0	12.975	62.2

T = test item; TC = toxicity control; R = reference substance; cumul. = cumulative ; U_r= relative uptake of oxygen in the water phase

Degradation Rates after 28 Days

Study Group	Oxygen Content after 0 d	Ur	BOD (mg O2)	% Degradation	Cumul. BOD (mg O2)	Cumul. Degradation (%)
T1	8.75	0.023	0.681	3.2	1.972	9.3
T2	8.70	0.011	0.343	1.7	1.127	5.7
TC	8.55	0.099	2.963	7.2	3.573	8.7
R1	8.75	0.034	1.022	4.9	16.139	77.3
R2	8.70	0.023	0.685	3.3	13.660	65.4

T = test item; TC = toxicity control; R = reference substance; cumul. = cumulative ; U_r= relative uptake of oxygen in the water phase

Validity criteria fulfilled:

yes

Remarks:

Percentage degradation of reference substance had reached the level of 60 % ThOD within 14 days and the total oxygen uptake in the blanks alter the first week of the test was lower than 3 mg O2/L and in the following week not ≥ 1 mg/L per week

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance degraded by 7.5% after 28 days. The test substance is not readily biodegradable.

Executive summary:

The study was conducted according to guidance of the German Environmental Agency (1990) (BODIS Test; similar to ISO 10708). Degradation of the test item was assessed over 28 days and a reference item, toxicity control and inoculum control were also included. Validity criteria were met. The toxicity control demonstrated toxic effects of the test substance to the microbial population of the test system. The study is not GLP-compliant bu the method is well described and it is therefore given a reliability score of 2 (reliable with restrictions). The test substance degraded by 7.5% after 28 days. The test substance is not readily biodegradable.

Description of key information

In both the key and supporting studies it was determined that the test item, is not readily biodegradable, with a maximum of 5 % and 7.5 % degradation, respectively.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The key study is GLP compliant and conducted in accordance with OECD guideline 301 D (2011). An inoculum control, functional control and toxicity control were all included. The validity criteria were met, apart from oxygen depletion in the inoculum control was 1.52 mg dissolved oxygen/L after 28 days, slightly above the 1.5 mg O2/L limit. However, this is not considered to have affected the integrity of the study. The study is considered to be reliable without restrictions (Klimisch 1). The test item reached maximum degradation of 5% after 14 days, and was shown to have degraded by 2% at 28 days. The test item is not readily biodegradable.

The supporting study was conducted according to guidance of the German Environmental Agency (1990) (BODIS Test; similar to ISO 10708) (1996). Degradation of the test item was assessed over 28 days and a reference item, toxicity control and inoculum control were also included. Validity criteria were met. The toxicity control demonstrated toxic effects of the test substance to the microbial population of the test system. The study is not GLP-compliant bu the method is well described and it is therefore given a reliability score of 2 (reliable with restrictions). The test substance degraded by 7.5% after 28 days. The test substance is not readily biodegradable.