Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

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:

12-December-2012-to-11-April-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

other: Sealed system, positive pressure basis, CO2-free air

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Activated Sludge was shaken to ensure homogeniety and allowed to settle until the overlying layer was clear. Samples of this supernatant were taken and used as the microbial inoculum.
- The sludge was not fed at any time during the study.
- Method of cultivation: Aeration until use.
- Storage conditions: Held within a 5L glass bottle and aerated for ca. 24h until test initiation.
- Preparation of inoculum for exposure: Sludge was allowed to settle before a portion of the overlying supernatant was siphoned off from the 5 L
sample to concentrate the solid sewage inoculant portion to the required level. The resultant sludge was then aerated and shaken by hand to
homogenise thoroughly. Sub-samples (5 mL) of the homogenised sludge were dried in an oven at approximately 105C and suspended solids content determined to be 3.80 g/L, which was within the protocol required range of 3-5 g/L. Sludge was aerated until use in test system.

Initial conc.:

31.8 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The test was conducted using 6 bioreactors (2 litre capacity), 2 each for control (Control 1 and Control 2) and test item (Test Item 1 and Test Item 2) and one each for the reference item (Reference) and toxicity control (Toxicity Control).

A totally sealed system was constructed and supplied, on a positive pressure basis, with CO2-free air. CO2 was removed by passing through self-indicating soda lime granules (Durasorb). The CO2-free air was passed through a glass manifold and subsequently through a secondary soda lime tube prior to entering each bioreactor. A non-return valve was placed in-line to prevent back-flow between the bioreactor and the first trap. The flow rate in the system was adjusted so the flow rate in each bioreactor was similar and in the protocol range of 30 100 mL/min; the recorded flow rates during the test were in the range 30-40 mL/min.
Addition of Additol® SXW 6246/90 coating additives to each test bioreactor and to the toxicity control bioreactor was effected via a glass cover slip.

A 1 g/L stock solution of the reference item was prepared by dissolving sodium benzoate (1.00009 g) in 1000 mL of mineral medium. Aliquots of this stock (69 mL) were added to each appropriate bioreactor to give an addition rate of 20 mg Dissolved Organic Carbon (DOC)/L.

To prepare the bioreactors, 1600 mL of mineral medium and 20 mL of microbial inoculum were added to each bioreactor and then purged with CO2-free air overnight. The appropriate weight of test item and/or volume of reference item stock solution were then quantitatively added to the appropriate bioreactors. Finally, and following pH measurement and adjustment if necessary, the bioreactors were made up to a final volume of 2000 mL with CO2-free mineral medium.

Each bioreactor was connected to 3 traps, each containing 100 mL of 0.0125M Ba(OH)2. At trap collection the trap closest to the bioreactor was taken for titration and the 2 remaining traps were moved one place closer to the bioreactor. A new trap containing 100 mL of 0.0125M Ba(OH)2 was then placed third in line from the bioreactor.

Trap changes and/or titrations were conducted on Days 2, 3, 5, 8, 10, 12, 16, 19, 23, 28 and 29. On Day 28, a trap collection was carried out as detailed above. The contents of each bioreactor were then acidified by the addition of 1 mL of concentrated hydrochloric acid to drive off any residual CO2 in the system; following overnight aeration, all traps were taken for analysis on Day 29.

Each sampled trap was titrated with 0.05M HCl; a few drops of phenolphthalein were added as the indicator, utilising a colour change from pink to colourless as the end point. The percent biodegradation was calculated from the titre.

The pH was determined in each bioreactor (Sentron Argus pH meter) on Days 0, (and adjusted to 7.2-7.6 with 1M HCl if necessary), 28 (pre-acidification) and 29.

Reference substance:

other: sodium benzoate-Fisher Scientific (Batch No. 0761409)

Parameter:

% degradation (CO2 evolution)

Value:

0.1

Sampling time:

2 d

Parameter:

% degradation (CO2 evolution)

Value:

0.5

Sampling time:

3 d

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

5 d

Parameter:

% degradation (CO2 evolution)

Value:

1.3

Sampling time:

8 d

Parameter:

% degradation (CO2 evolution)

Value:

>= 1.3

Sampling time:

10 d

Parameter:

% degradation (CO2 evolution)

Value:

1.6

Sampling time:

12 d

Parameter:

% degradation (CO2 evolution)

Value:

1.7

Sampling time:

16 d

Parameter:

% degradation (CO2 evolution)

Value:

1.7

Sampling time:

19 d

Parameter:

% degradation (CO2 evolution)

Value:

1.9

Sampling time:

23 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

2.2

Sampling time:

28 d

Details on results:

The substance coating additives, at a nominal addition rate of 20 mg TOC/L (31.8 mg test item/L), was not ready biodegradable under the conditions of the test since a transition from 10% to 60% biodegradation in a 10 day window during the 28 day test was not achieved. The mean cumulative biodegradation of the test item at Day 28 was 2.2%.

The TOC content of the toxicity control bioreactor was equivalent to the combined TOC of the individual test and reference bioreactors. The cumulative biodegradation observed for the toxicity control expressed in terms of the total TOC added was 52.0% which correlated well with the mean (48.0%) cumulative biodegradation for the separate test (2.2% (mean)) and reference (93.8%) item data. This close correlation indicates that the test item was not inhibitory to the microbial inoculum under test conditions.

The test met the following guideline validity criteria:

• The mean CO2 evolution in the controls was <40 mg/L (mean = 10.2 mg/L) at Day 28.
• The reference item achieved 60% biodegradation by Day 14.

The difference in extremes of the replicate test bioreactors was 38.9% which exceeds the 20% limit specified in the Test Guideline (Reference 1) for a valid test. The evolution of CO2 from the test bioreactors was low resulting in low individual cumulative biodegradation values of 2.5 (Test 1) and 1.8 (Test 2) at Day 28; the magnitude of these data are judged to be the reason why the test failed to satisfy this validity criterion.

The test is considered to be valid.

Results with reference substance:

The reference item was readily biodegradable under the conditions of the test as >60% biodegradation was achieved by Day 5; this demonstrates the acceptable viability of the inoculum.

The results of the test were as follows (28 day percent biodegradation values):

% Biodegradation	The substance coating additives (Mean)	Sodium Benzoate	Toxicity Control
			A	B
	2.2	93.8	104.2	52.0
The cumulative biodegradation determined in the Toxicity Control bioreactor is expressed in terms of the TOC content resulting from the reference item only (A) and for the TOC resulting from the reference and test item (B).

Trap Sampling and Titre Volumes (0.05M HCl) :

Day No.	Background Titre (mL)	Bioreactor
		Control 1 (mL)	Control 2 (mL)	Test Item 1 (mL)	Test Item 2 (mL)	Reference Item (mL)	Toxicity Control (mL)
2	51.37	49.08	48.26	48.49	48.56	15.59	16.46
3	51.37	49.90	49.41	48.95	49.11	21.74	22.33
5	51.37	50.10	49.37	48.88	49.31	26.87	24.30
8	51.27	49.71	49.38	48.85	49.41	35.06	32.32
10	51.11	49.72	50.13	49.98	50.19	41.44	40.53
12	51.10	50.44	50.50	50.26	49.84	45.19	44.99
16	50.81	50.31	50.15	49.89	50.27	44.33	43.95
19	51.80	50.11	49.99	50.43	50.27	46.85	44.93
23	51.33	50.12	50.21	49.85	49.84	47.68	45.51
28	51.12	49.21	49.16	49.15	48.81	47.81	44.91
29*	51.49	50.00	50.11	49.67	50.13	49.48	48.15
29*	50.95	49.94	49.91	50.01	50.03	49.93	49.71
29*	51.17	50.04	50.09	50.38	50.45	50.07	50.04
* = The biodegradation process was terminated following Day 28 analysis and trap change; all traps remaining were taken for analysis on Day 29

Cumulative Biodegradation (%)

Day No.	Test Item: The substance coating additives		Reference Item: Sodium Benzoate	Toxicity Control: Test and Reference Items
	Replicate 1	Replicate 2		A	B
2	0.1	0.1	24.8	24.1	12.1
3	0.6	0.5	45.7	44.5	22.3
5	1.2	0.8	62.9	63.6	31.8
8	1.7	0.9	73.7	76.5	38.2
10	1.7	0.9	80.0	83.5	41.7
12	1.8	1.4	83.9	87.6	43.7
16	2.0	1.4	88.3	92.2	46.0
19	2.0	1.4	90.6	96.0	47.9
23	2.2	1.6	92.4	99.5	49.6
28*	2.5**	1.8**	93.8	104.2	52.0
*   A trap change was conducted on Day 28 after which the biodegradation process was terminated and a final trap analysis conducted on Day 29; the tabulated result represents the sum of all analyses   ** Mean cumulative biodegradation for Test Bioreactors = 2.2%   The cumulative biodegradation determined in the Toxicity Control bioreactor is expressed in terms of the total organic carbon resulting from the reference item only (A) and for the total organic carbon resulting from the reference and test items (B).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance was not ready biodegradable under the conditions of the test. A mean total of 2.2% degradation was achieved by Day28.

Executive summary:

A study was conducted to determine the ready biodegradability of according to the OECD Guideline 301 B (CO2-evolution test method), in compliance with GLP. The test was conducted at a nominal loading rate of 20 mg Total Organic Carbon (TOC)/L (31.8 mg of the test substance/L). A total of 6 bioreactors (2 litre capacity) were used: 2 each for the test substance and control and one each for the reference substance (sodium benzoate) and toxicity control (the test substance and sodium benzoate). The reference substance was introduced into the respective bioreactor dissolved in mineral medium at a level to provide a final concentration of 20 mg Dissolved Organic Carbon (DOC)/L. Levels of CO2 evolved were determined by titrating the contents of the Ba(OH)2 traps against 0.05 M HCl using phenolphthalein indicator.

The results of the test were as follows (28 day percent biodegradation values):

% Biodegradation	the substance coating additives (Mean)	Sodium Benzoate	Toxicity Control
			A	B
	2.2	93.8	104.2	52.0
The cumulative biodegradation determined in the Toxicity Control bioreactor is expressed in terms of the TOC content resulting from the reference item only (A) and for the TOC resulting from the reference and test item (B).

The test substance was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10-day window during the 28-day test. The mean cumulative biodegradation of the test item at Day 28 was 2.2%. The test substance was not considered to be inhibitory to the microbial inoculum since the biodegradation observed in the toxicity control bioreactor was similar to that expected from the individual test and reference bioreactors. The reference substance showed 93.8% degradation after 28 days. Regarding the validity criteria, the test fulfilled all two out of three validity criteria, i.e. (1) the mean CO2 evolution in the controls was <40 mg/L (mean = 10.2 mg/L) at Day 28; (2) and the reference substance achieved 60% biodegradation by Day 14. For the third criteria, the difference in extremes of the replicate test bioreactors was 38.9% which exceeded the 20% limit. The evolution of CO2 from the test bioreactors was low resulting in low individual cumulative biodegradation values of 2.5 (Test 1) and 1.8 (Test 2) at Day 28; the magnitude of these data was judged to be the reason why the test failed to satisfy the 3^rdvalidity criterion. Overall, the test was considered to be valid. Under the study conditions, the test substance is considered to be not readily biodegradable ( Hall, 2013).

Description of key information

The test substance was not readily biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of according to the OECD Guideline 301 B (CO2-evolution test method), in compliance with GLP. The test was conducted at a nominal loading rate of 20 mg Total Organic Carbon (TOC)/L (31.8 mg of the test substance/L). A total of 6 bioreactors (2 litre capacity) were used: 2 each for the test substance and control and one each for the reference substance (sodium benzoate) and toxicity control (the test substance and sodium benzoate). The reference substance was introduced into the respective bioreactor dissolved in mineral medium at a level to provide a final concentration of 20 mg Dissolved Organic Carbon (DOC)/L. Levels of CO2 evolved were determined by titrating the contents of the Ba(OH)2 traps against 0.05 M HCl using phenolphthalein indicator. The test substance was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10-day window during the 28-day test. The mean cumulative biodegradation of the test substance at Day 28 was determined to be 2.2%. The test substance was not considered to be inhibitory to the microbial inoculum since the biodegradation observed in the toxicity control bioreactor was similar to that expected from the individual test and reference bioreactors. The reference substance showed 93.8% degradation after 28 days. Regarding the validity criteria, the test fulfilled two out of three validity criteria, i.e. (1) the mean CO2 evolution in the controls was <40 mg/L (mean = 10.2 mg/L) at Day 28; (2) and the reference substance achieved 60% biodegradation by Day 14. For the third criteria, the difference in extremes of the replicate test bioreactors was 38.9% which exceeded the 20% limit. The evolution of CO2 from the test bioreactors was low resulting in low individual cumulative biodegradation values of 2.5 (Test 1) and 1.8 (Test 2) at Day 28; the magnitude of these data was judged to be the reason why the test failed to satisfy the 3rdvalidity criterion. Overall, the test was considered to be valid. Under the study conditions, the test substance is considered to be not readily biodegradable ( Hall, 2013).