Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 November 2013 and 4 January 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 301D using a closed bottle method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Molecular formula:

C10H14O2

IUPAC Name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Test material form:

other: Liquid

Details on test material:

Test substance: Bicyclo[2.2.1]hept-5-ene-2-carboxylic acid, ethyl ester
Test substance code: FRET 07-0117
CAS No.: 10138-32-6
Molecular formula: C10H14O2
Formula weight: 166

Physicochemical properties
Appearance: Liquid
Water solubility 840 mg/L (20 °C)
Vapor pressure 35.4 Pa/0.266 mm Hg
Stability Stable in water
Storage
Storage condition Store at room temperature and keep in dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Information on inoculum
The secondary effluent used as the inoculum in this test was collected from the Wuzhong district urban area sewage treatment plant. The address of the plant is No. I, Baodai East Road, Suzhou city.
The batch No. of inoculum was SE-20131118-01.

Justification for selection of inoculum
The secondary effluent from a domestic waste water treatment plant is recommended by test guideline.

Pre-treatment of inoculum
The secondary effluent was let to stand for I hour, after which the supernatant was poured off supernatant and the secondary effluent was maintained aerobic at the test temperature.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test conditions
Temperature: 20 ± 1 °C
Photoperiod: dark
Test container: 125 mL BOD bottles
Duration: 28 days

Methods
Exposure method and Justification
According to the information on the test substance and the test guideline, the ready biodegradability of the test substance will be determined using the closed bottle test method. Since the solubility of the test substance FRET07-0117 is 840 mg/L (20 °C), the test solution will be prepared from a stock solution of the test substance.

Justification for test design
According to OECD test guideline 301D, the added volume of the inoculum is in the range of 0.05 mL to 5 mL per liter of medium. Moreover, the ThOD values for sodium benzoate and the test substance were calculated to be 1.67 mg 0 2 /mg sodium benzoate and 2.41 mg 0 2 /mg of test substance, respectively. For a test to be valid the saturated oxygen concentration in water should be about 9 mg/L.
The final test concentrations for both the reference substance (sodium benzoate) and the test substance (Bicyclo[2.2. l]hept-5-enc-2-carboxylic acid, ethyl ester ) were 2 mg/L and the final inoculum concentration was 0.2 mL/L.

Test medium
The medium was strongly aerated for about 4 hours after preparation and was ready for use after standing for 22 h. The concentration of dissolved oxygen was 9.48 mg/L.

Preparation of test solutions
Inoculum blank
The fully-aerated medium was transferred to a test flask so that they were about one-third full and then inoculated with 1 mL of secondary effluent. The test solution was made up a final volume of 5 litres with aerated mineral medium using a hose which reached down to the bottom of the bottle to achieve adequate mixing.

Procedure control
Stock solution: weighed 0.2001 g sodium benzoate and dissolved in mineral medium and made up to 100 mL. Fully-aerated medium was added to test flask to about one-third full, and then 5 mL of the stock solution of the reference substance was added. This solution was inoculated with 1 mL of secondary effluent, and then made up to a final volume of 5 litres with aerated mineral medium using a hose which reached down to the bottom of the bottle to achieve adequate mixing.

Test suspension
Stock solution: weighed 0. 0503 g test substance and dissolved in mineral medium and made up to 500 mL. Fully-aerated medium was added to a test flask to about one-third full and then added 100 mL stock solution of test substance. This solution was inoculated with 1 mL of secondary effluent and made up to a final volume of 5 litres with aerated mineral medium using a hose which reached down to the bottom of the bottle to achieve adequate mixing.

Toxicity control
Fully-aerated medium was added to a test flask to about one-third full and then 40 mL of the test substance stock solution was added along with 2 mL of reference substance stock solution of reference substance. This solution was inoculated with 0.4 mL of secondary effluent then made up to a final volume of 2 litres with aerated mineral medium using a hose which reached down to the bottom of the bottle to achieve adequate mixing.

Incubation
Each prepared solution was dispensed into the respective group of BOD bottles by hose from the lower quarter (not the bottom) of the appropriate large bottle, so that all the BOD bottles were completely filled. Each bottle was tapped gently to remove any air bubbles and incubated at the test temperature in the dark.

Sampling and analysis
On days 0, 3, 7, 11, 14, 17, 21, 24 and 28, the dissolved oxygen concentration was analyzed for the test suspension, procedure control and inoculum blank. At days 0, 7 and 14, the concentration of dissolved oxygen was analysed for the toxicity control.

Date treatment
ThOD calculation
The theoretical oxygen demand (ThOD) was calculated by the formula below. For the compound:
CcHhClclNnNanaOoPpSs, the ThOD, without nitrification, would be:

ThOD = (16[2c + ½ (h – cl – 3n) + 3s + 5/2p + ½ na – o] mg/mg)/ MW

According to the formula above and the information of the test substance and reference substance, the ThOD of test substance is 2.41 mg/mg, and the ThOD of the reference substance is 1.67 mg/mg.

Biodegradation
The percent biodegradation for the test substance will be calculated according to the following formula:

% Degradation = ((BOD (mgO2 / mg chemical)) / ThOD (mgO2 / mg chemical)) x 100

Where:
BOD= Biochemical oxygen demand of the test substance

= ((mgO2 update / L by test substance) – (mg O2 uptake / L by inoculum control) / mg substance per litre
= mgO2 /mg test substance

Statistics and calculation will be performed using in MS EXCEL 2010.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

10.17

Sampling time:

28 d

Remarks on result:

other: Mean of duplicate samples

Details on results:

The percent biodegradation of the test substance was measured in duplicate and the values were 9.75% and 9.96% (mean 9.85%) on day 14, and 10.06% and 10.27% (mean 10.17%) at the end of the test.
The mean percent biodegradation of the toxicity control and the procedure control values were 31.56% and 71.56% respectively on day 14. These results demonstrated that the inoculum was not inhibited by the test substance and that the activity of inoculum met the test requirements. The mean percent biodegradation value for the toxicity control was 80.39% on day 28.
The biodegradation curve was plotted as the mean percent biodegradation during the test period.

BOD5 / COD results

Results with reference substance:

The mean percent biodegradation of the toxicity control and the procedure control values were 31.56% and 71.56% respectively on day 14.

Any other information on results incl. tables

Temperature

The temperature during the test period was in the range of 19.4 - 20.5°C.

ThOD in groups

Substance name	ThOD (mg/mg)	Test suspension		Procedure control		Toxicity control
		Test Conc. (mg/L)	ThOD (mg/L)	Test Conc. (mg/L)	ThOD (mg/L)	Test Conc. (mg/L)	ThOD (mg/L)
FRET 07-0117	2.41	2	4.82	0	3.34	2	8.16
Sodium benzoate	1.67	0		2		2

 

Concentration of dissolved oxygen during the test

Group	Flask No.	Concentration of dissolved oxygen at day N (mg/L)
		N = 0	N = 3	N = 7	N = 11	N = 14	N = 17	N = 21	N = 24	N = 28
Inoculum blank	1	9.47	9.18	9.33	9.36	9.40	9.18	9.05	9.24	9.23
	2	9.43	9.17	9.36	9.28	9.38	9.14	9.00	9.29	9.14
	Average	9.45	9.18	9.35	9.32	9.39	9.16	9.03	9.27	9.19
Test suspension	1	9.35	9.12	8.98	8.52	8.82	8.65	8.43	8.57	8.60
	2	9.40	9.10	8.93	8.68	8.86	8.69	8.43	8.63	8.64
Procedure control	1	9.47	7.68	7.07	7.13	7.01	6.82	6.51	6.70	6.26
	2	9.38	7.64	7.13	6.95	6.94	6.88	6.58	6.67	6.69
Toxicity control	1	9.43	-	6.78	-	6.74	-	-	-	-
	2	9.40	-	6.73	-	6.82	-	-	-	-

- not available

 

Oxygen depletion and biodegradation rate

Name	Flask No.	Day N
		N = 0	N = 3	N = 7	N = 11	N = 14	N = 17	N = 21	N = 24	N = 28
Average oxygen depletion of inoculum blank (mg/L)	-	0.00	0.27	0.11	0.13	0.06	0.29	0.42	0.18	0.26
Oxygen depletion of procedure control (mg/L)	1	0.00	1.79	2.40	2.34	2.46	2.65	2.96	2.77	3.21
Biodegradation rate of procedure control (%)	2	0.00	1.74	2.25	2.43	2.44	2.50	2.80	2.71	2.69
Oxygen depletion of test suspension (mg/L)	1	0.00	45.36	68.71	66.17	71.86	70.66	75.90	77.40	88.17
	2	0.00	43.86	64.22	68.86	71.26	66.17	71.11	75.60	72.60
	Average	0.00	44.61	66.47	67.51	71.56	68.41	73.50	76.50	80.39
Biodegradation rate of test suspension (%)	1	0.00	0.23	0.37	0.83	0.53	0.70	0.92	0.78	0.75
	2	0.00	0.30	0.47	0.72	0.54	0.71	0.97	0.77	0.76
Biodegradation rate of test suspension (%)	1	0.00	-0.93	5.50	14.52	9.75	8.51	10.27	12.34	10.06
	2	0.00	0.52	7.57	12.24	9.96	8.71	11.31	12.14	10.27
	Average	0.00	-0.21	6.54	13.38	9.85	8.61	10.79	12.24	10.71
Oxygen depletion of toxicity control (mg/L)	1	0.00	-	2.65	-	2.69	-	-	-	-
	2	0.00	-	2.67	-	2.58	-	-	-	-
Biodegradation rate of toxicity control (%)	1	0.00	-	31.19	-	32.23	-	-	-	-
	2	0.00	-	31.43	-	30.88	-	-	-	-
	Average	0.00	-	31.31	-	31.56	-	-	-	-

- not available

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The mean percent biodegradation of test substance FRET 07-0117 was 10.17% at the end of the test under the current test conditions.

Executive summary:

The ready biodegradability of the test substance, TM 09-218, was assessed according to OECD Test Guideline 301D using a closed bottle method. The mean percent biodegradation of test substance was 10.17% at the end of the test.