2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

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:

18 August 2009 to 15 October 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 301B Ready Biodegradability CO2 Evolution Test. Beijing: China Environmental Science Press.

Version / remarks:

2004.337-343. First edition

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China The Guidelines for the test of chemical (HJ/T 153-2004). Beijing: China Environmental Science Press.

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: (4) State Environmental Protection Administration of China The Guidelines for the hazard evaluation of new chemical substances (HJ/T 154-2004). Beijing:China Environmental

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: the inoculum was derived from the secondary effluent of a treatment plant receiving dominantly domestic sewage in Guangzhou, which is belonged to the Da Tansha Sewage Treatment Plant.
- Pre-treatment: the sludge was removed of any coarse particles and impurities on the surface, then was washed with the mineral medium, and was kept aerobic until use.
- Concentration of sludge: Measured concentration of the sludge was 5.0 g/L. The inoculum was diluted with mineral medium to yield a concentration of 4.0 g suspended solids/L. 15 mL inoculum was inoculated in each flask to yield a final concentration of 30 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Test Water: Deionised water.
Test Medium:
(a) 8.5 g potassium dihydrogen phosphate, 21.75 g potassium hydrogen phosphate, 33.4 g second hydrated disodium hydrogen phosphate and 0.5 g ammonium chloride. Dissolve in water and make up to 1 L. The pH of the solution should be 7.4
(b) 27.5 g anhydrous calcium chloride dissolved in water and made up to 1 L
(c) 22.5 g magnesium sulphate 7 hydrate dissolved in water and made up to 1 L.
(d) 0.25 g six hydrated ferric chloride dissolved in water and made up to 1 L.

-10 mL of solution (a) was mixed with 800 mL deionised water, then 1 mL of solutions (b), (c) and (d) were added and made up to 1 L with water.
- Test temperature: 22 ± 2°C
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Flasks, 3 litres, each fitted with an aeration tube reaching nearly to the bottom of the vessel and an outlet
- Number of culture flasks/concentration: Flasks 1 and 2 contained test material and inoculum (test suspension); Flasks 3 and 4 contained only inoculum (inoculum blank), Flask 5 contained reference compound and inoculum (procedure control) and Flask 6 contained test material, reference compound and inoculum (toxicity control).
- Method used to create aerobic conditions: by bubbling CO2-free air through the suspensions at a rate of 30 mL/min -100 mL/min.

- Measuring equipment:
-Three absorption bottles, each containing 100 mL of 0.0125 mol/L barium hydroxide solution, were connected in series to each 3-litre flask. The solution must be free of precipitated sulphate and carbonate and its strength was determined immediately before use.
- The Barium hydroxide absorption method: on the days of CO2 measurement, the barium hydroxide absorber closest to the test vessel was disconnected and the hydroxide solution was titrated with 0.05 mol/L HCI using phenolphthalein as the indicator. The remaining absorbers were moved one place closer to the test vessel and a new absorber containing 100 mL fresh barium hydroxide was placed at the far end of the series. On day 28, 1 mL of concentrated hydrochloric acid was added to each test vessel and the test suspensions were aerated overnight to drive off the carbon dioxide present in the test suspensions. On day 29 the last analysis of evolved carbon dioxide was made.

SAMPLING
- During the first ten days, analyses of CO2 were made every second or third day and then at least every fifth day until the 28th day (the analyses are made until 14 day in the process control and the toxicity control).

CONTROL AND BLANK SYSTEM
- Inoculum blank: Flasks 3 and 4 containing only inoculum
- Procedure control: Flask 5 containing reference compound and inoculum (procedure control)
- Toxicity control: Flask 6: containing test material, reference compound and inoculum

DATA PROCESSING
-The amount of CO2 produced was calculated from the amount of base remaining in the absorption bottles.
-The graph of percentage degradation against time for the test and reference substances were drawn, to indicate the 10 day window. If possible, the percentage removal at plateau, at the end of the test and/or after 10 day window were calculated and reported.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 2.6 - <= 15

Sampling time:

28 d

Details on results:

-Percentage degradation of the test material was 1.2 and 5.4% at the 10th day and was 2.6 and 15% at the end of the test. Results can be seen in Table 1.
-Percentage degradation of toxicity control was 37% at day 14.

QUALITY ASSURANCE
-The total CO2 evolution in the inoculum blank at the end of the test did not exceed 70 mg/L medium.
-The percentage degradation (based on total ThOD or ThCO2) of the reference compound had reached the pass levels of 60% by day 14.
-The difference of extremes of replicate values of the removal of the test material at the plateau, at the end of the test or at the end of the 10 day window was <20%.
-In a toxicity test, more than 25% degradation (based on total ThOD or ThCO2) occurred within 14 days.

Results with reference substance:

The percentage degradation of the reference substance Sodium Benzoate was 96%.

Table 1: Cumulative CO₂ produced and percentage degradation during the test

Time (d)	Cumulative CO2 production (mg)				Cumulative degradation rate (%)
	Flask 1 (Test suspension)	Flask 2 (Test suspension)	Flask 5 (Procedure control)	Flask 6 (Procedure control)	Flask 1 (Test suspension)	Flask 2 (Test suspension)	Flask 5 (Procedure control)	Flask 6 (Procedure control)
2	0.00	0.00	32.1	19.7	0.0	0.0	36	11
4	0.00	0.00	50.1	35.0	0.0	0.0	57	20
7	0.00	1.49	62.1	44.6	0.0	1.7	70	25
10	1.09	4.75	70.0	52.1	1.2	5.4	79	30
14	1.80	7.87	84.8	64.9	2.0	9.0	96	37
17	1.88	9.34	-	-	2.1	11	-	-
20	1.88	9.93	-	-	2.1	11	-	-
24	2.25	13.1	-	-	2.6	15	-	-
28	2.25	13.3	-	-	2.6	15	-	-

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of the study the test material was not readily biodegradable.

Executive summary:

The ready biodegradability of the test material was determined in accordance with the standardised guideline OECD 301B and other Chinese guidelines under GLP conditions. An aerobic aqueous test system was used to assess the biodegradability of the test material. A measured volume of inoculated mineral medium, containing 18 mg/L of the test material (corresponding to 12 mg TOC/L) as the nominal sole source of organic carbon, was aerated by the passage of CO₂-free air at a controlled rate in the dark or in diffuse light. Degradation was followed over 28 days by determining the CO₂ produced using the barium hydroxide absorption method. The test suspension and inoculum blanks were performed in duplicate and single vessels were used for the procedure and toxicity controls.

Under the conditions of the study, percentage degradation of reference substance sodium benzoate was 96% at day 14. Since all criteria for acceptability of the test were met, this study was considered to be valid. Percentage degradation of test material was 1.2 and 5.4% at day 10 and 2.6 and 15% at the end of the test. Percentage biodegradation of the test material was < 60% of ThCO₂ production within the first 10 days over a 28 day test period. Under the conditions of the study the test material was not readily biodegradable.

Description of key information

Under the conditions of the study the test material was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test material was determined in accordance with the standardised guideline OECD 301B and other Chinese guidelines under GLP conditions. An aerobic aqueous test system was used to assess the biodegradability of the test material. A measured volume of inoculated mineral medium, containing 18 mg/L of the test material (corresponding to 12 mg TOC/L) as the nominal sole source of organic carbon, was aerated by the passage of CO₂-free air at a controlled rate in the dark or in diffuse light. Degradation was followed over 28 days by determining the CO₂ produced using the barium hydroxide absorption method. The test suspension and inoculum blanks were performed in duplicate and single vessels were used for the procedure and toxicity controls.

Under the conditions of the study, percentage degradation of reference substance sodium benzoate was 96% at day 14. Since all criteria for acceptability of the test were met, this study was considered to be valid. Percentage degradation of test material was 1.2 and 5.4% at day 10 and 2.6 and 15% at the end of the test. Percentage biodegradation of the test material was < 60% of ThCO₂production within the first 10 days over a 28 day test period. Under the conditions of the study the test material was not readily biodegradable.