Ammonium 2-ethylhexyl sulphate

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:

2020-05-12 to 2020-06-17

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:

Adopted on 17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

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: the sewage plant for domestic sewage in Balatonfüred, Hungary
- Storage conditions: The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 °C
- Storage length: 6 days
- Pretreatment: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
- Concentration of sludge: 5 g dry material per litre
- Initial cell/biomass concentration: 1E7 - 1E9 cells/L

Duration of test (contact time):

28 d

Initial conc.:

24 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: prepared according to study guideline
- Test temperature: 22 ± 2 °C (20.0 - 21.4 °C)
- pH: 7.4 ± 0.2
- pH adjusted: yes, with 1N HCl
- Cell biomass: 100,000,000 cells/L
- Aeration of dilution water: yes (2 L/minute)
- Suspended solids concentration: ~25 mg suspended solid/L (≤ 30 mg suspended solids/L)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Large glass tank (volume: ~ 30 L)
- Number of culture flasks/concentration: 7
- Method used to create aerobic conditions: Gas cylinder providing CO2-free oxygen and CO2-free nitrogen mixture (20 % O2: 80 % N2)
- Measuring equipment: Carbon analyser
- Test performed in closed vessels due to significant volatility of test substance: yes, but test substance is not volatile

SAMPLING
Measurement of the evolved CO2 in all test groups (test item, inoculum control, procedure control, abiotic control and toxicity control) was performed on the 2nd, 6th, 7th, 8th, 10th, 14th, 17th, 20th, 28th days and the last analysis was performed on test day 29.
On the days of the CO2 measurements the barium hydroxide absorption bottle closest to the test flask was disconnected and titrated with 0.05 M HCl to measure the remaining base content in presence of phenolphthalein until the first indicator color change (pH ≤ 8.2). The remaining absorption bottles were placed one or two positions closer to the test flask, and new absorption bottle(s) containing 100 mL fresh 0.0125 M Ba(OH)2 were attached at the far end of the series.
Due to the intensive CO2 evolution in the test item, procedure and toxicity controls, the content of additional absorption bottles from the attached series was titrated in these groups. The analyzed bottles were replaced with new absorption bottles containing 100 mL fresh 0.0125 M Ba(OH)2.
On test day 28, samples (about 20 mL from the test item containing flasks and inoculum control) were taken from the test flask for DOC analysis and for viability checking of the test mixtures and sterility checking of abiotic control, thereafter 1 mL concentrated HCl was added to all flasks and aerated overnight to drive off the CO2 present in the test suspension.
One absorption bottle was left attached to each test flask. On day 29 the last CO2 analysis was performed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Test item group: 2
- Abiotic control: 1
- Procedure control: 1
- Toxicity control: 1

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

70.19

Sampling time:

28 d

Details on results:

The percentage biodegradation of the test item reached a mean of 62.18 % (based on its theoretical carbon dioxide, ThCO2) on the 17th day of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item (derived from the biodegradation curve) was reached on about the 7th day of the test and the biodegradation reached the 60 % level between the 16th and 17th days of test. The pass level, 60 % of ThCO2 was reached in about 9 - 10 days immediately following the attainment of 10 % biodegradation. The 10-day window was estimated from the evolved CO2 determinations and the values were additionally derived from the biodegradation curve. Based on the obtained values the test item is considered to be readily biodegradable.

Results with reference substance:

Based on its ThCO2 the reference item Sodium benzoate was sufficiently degraded to 66.88 % already after 10 days, and to 78.87 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum.

Table 1: The Calculated Weight of Produced CO₂

 

Test group	Flask number	The Calculated Weight of Evolved CO2 after n days of exposure (mg)
		2	6	7	8	10	14	17	20	28	28	29
Test item group	1						7.) 15.85	9.) 24.67	11.) 21.30
		1.)   2.59	2.)   8.64	3.) 13.23	4.) 20.96	5.) 32.65	6.) 49.52	8.) 51.61	10.) 52.65	12.) 48.75	13.) 24.97	14.)   8.07
	Sum:	2.59	8.64	13.23	20.96	32.65	65.37	76.29	73.94	48.75	24.97	8.07
	2						7.) 13.75	9.) 23.69	11.) 26.17
		1.)   1.38	2.) 13.28	3.) 14.91	4.) 18.84	5.) 28.41	6.) 50.18	8.) 50.71	10.) 53.94	12.) 50.68	13.) 27.86	14.)   9.67
	Sum:	1.38	13.28	14.91	18.84	28.41	63.93	74.40	80.11	50.68	27.86	9.67
Mean		1.98	10.96	14.07	19.90	30.53	64.65	75.34	77.03	49.71	26.42	8.87
Inoculum control	3	1.)   2.71	2.)   3.98	3.)   1.84	4.)   1.54	5.)   1.94	6.)   3.22	7.)   5.29	8.)   2.41	9.)   3.58	10.)   1.28	11.)   2.32
	4	1.)   2.21	2.)   1.60	3.)   3.38	4.)   5.64	5.)   6.24	6.)   4.03	7.)   6.61	8.)   2.51	9.)   4.25	10.)   2.82	11.)   1.22
Mean		2.46	2.79	2.61	3.59	4.09	3.62	5.95	2.46	3.91	2.05	1.77
Sum of Evolved CO2:		35.29
Procedure control	5				5.) 11.79	7.) 23.22	9.) 26.93	11.) 36.75	13.) 38.36
		1.) 10.01	2.) 37.04	3.) 49.43	4.) 50.82	6.) 54.46	8.) 54.03	10.) 52.71	12.) 49.37	14.) 53.03	15.) 32.33	16.)   9.15
	Sum:	10.01	37.04	49.43	62.61	77.68	80.96	89.46	87.73	53.03	32.33	9.15
Abiotic control	6	1.)   1.33	2.)   1.01	3.)   2.66	4.)   5.29	5.)   8.25	6.)   12.50	7.)   14.74	8.)   15.74	9.)   14.64	10.)   10.47	11.)   4.68
Toxicity control	7				5.) 14.73	7.) 38.35	9.) 46.37	11.) 51.55	13.) 50.35
		1.)   9.87	2.) 35.88	3.) 49.36	4.) 53.97	6.) 55.10	8.) 54.67	10.) 52.70	12.) 55.57	14.) 54.07	15.) 42.81	16.) 28.45
	Sum:	9.87	35.88	49.36	68.70	93.45	101.04	104.25	105.92	54.07	42.81	28.45

 

Remark: The serial numbers of 1.), 2.), 3.) to 16.) indicate the number of titrated absorption bottle.

Remark: The weight of CO₂ produced (mg) was calculated as: 1.1 x (50 mL HCl titrated), where 50 is a theoretical value: 50 mL 0.05 M HCl is needed to titrate 100 mL 0.0125 M Ba(OH)_2.

 

 

Table 2: The Calculated Weight of Produced CO₂ corrected with Inoculum Values

Test group	Flask number	The Calculated, Corrected Weight of Evolved CO2 after n days of exposure (mg)
		2	6	7	8	10	14	17	20	28
Test item group	1	0.13	5.85	10.63	17.36	28.56	61.75	70.33	71.48	74.07
	2	-1.08	10.49	12.30	15.25	24.33	60.31	68.45	77.65	80.48
Mean		-0.48	8.17	11.46	16.31	26.44	61.03	69.39	74.57	77.28
Procedure control	5	7.55	34.25	46.83	59.02	73.60	77.34	83.51	85.27	86.78
Abiotic control	6	1.33	1.01	2.66	5.29	8.25	12.50	14.74	15.74	29.79
Toxicity control	7	7.41	33.09	46.75	65.10	89.36	97.41	98.30	103.46	117.60

 

Remark: For the procedure control and toxicity control more absorption bottles were titrated at each occasion. The calculated evolved CO₂ values were summed, thereafter corrected with the corresponding inoculum value. For non-corrected “calculated weight of produced CO₂” values, please refer to Table 1.

The abiotic control values were not corrected with the corresponding inoculum value.

 

 

Table 3: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days Calculated Based on ThCO₂

Test group	Flask number	Percentage Biodegradation after n days of exposure (%)
		2	6	7	8	10	14	17	20	28
Test item group	1	0.11	5.24	9.52	15.56	25.59	55.33	63.02	64.05	66.37
	2	-0.97	9.40	11.02	13.67	21.80	54.04	61.34	69.58	72.12
Mean		-0.43	7.32	10.27	14.61	23.70	54.68	62.18	66.82	69.24
Procedure control	5	6.86	31.12	42.55	53.64	66.88	70.28	75.89	77.49	78.87
Abiotic control	6	1.19	0.91	2.39	4.74	7.39	11.20	13.21	14.10	26.69
Toxicity control	7	3.34	14.93	21.09	29.37	40.32	43.95	44.35	46.68	53.06

 

Remark:   The percentage biodegradation values are calculated according to the following equation:

% biodegradation = (mg CO₂ produced)/(ThCO₂ x mg test substance added)×100

Where the produced amount of CO₂ is originated from the calculated weight of produced CO₂ in table 1, the ThCO₂ of the test item is 1.55 mg CO₂ / mg test item, and the test flask (with 3 L reaction mixture) contained 3 x 24 mg (with purity corrected amount) = 72 mg test item/ flask.

Where the produced amount of CO₂ is originated from the calculated weight of produced CO₂ in table 1, the ThCO₂ of the reference item is 2.14 mg CO₂ / mg reference item, and the test flask (with 3 L reaction mixture) contained 3 x 17.14 mg = 51.42 mg reference item/ flask.

 

Table 4: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days Calculated Based on TOC (after DOC Analysis)

Test group	Flask number	Percentage Biodegradation after n days of exposure (%)
		2	6	7	8	10	14	17	20	28
Test item group	1	0.11	5.31	9.65	15.77	25.94	56.08	63.88	64.93	67.27
	2	-0.98	9.53	11.17	13.85	22.09	54.78	62.17	70.53	73.10
Mean		-0.43	7.42	10.41	14.81	24.02	55.43	63.03	67.73	70.19
Procedure control	5	6.86	31.11	42.53	53.61	66.84	70.24	75.85	77.44	78.82
Abiotic control	6	1.21	0.92	2.42	4.81	7.49	11.35	13.39	14.30	27.06
Toxicity control	7	3.36	15.03	21.23	29.57	40.58	44.24	44.64	46.98	53.40

 

Remark: The percentage biodegradation values are calculated according to the following equation:

% biodegradation = (mg CO₂ produced)/(mgTOC added in the test × added)×100

Where the produced amount of CO₂ is originated from the Table 2, the TOC (that was considered to be equal with DOC) of the test item and reference item is 10 mg/L (nominal), (consequently 20 mg/L in the toxicity control, containing both, the test and reference items) and the test flask contained 3L reaction mixture.

 

Validity of the Study

The study is considered valid because:

- the IC content of the test item stock solution suspension in the mineral medium at the beginning of the test (0.74 %) was lower than 5 % of the total carbon (TC).

- the total CO₂ evolution in the inoculum blank at the end of the test (on average 35.29 mg, corresponding to 11.76 mg/L medium) did not exceed 40 mg/L medium.

- the difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %: in the test item group the differences between the duplicate biodegradability values from the 17th to 28th day of the test (at about the biodegradation plateau) were 2.7 and 8.3 %.

- the percentage degradation of the reference item reached the level of ready biodegradability (> 60 %) on exposure day 10. The percentage degradation of the reference item was 70.28 % on day 14.

- in the toxicity control, containing both the test item and reference item the biodegradation was higher than 25 % based on ThCO2 on day 14. The biodegradation in the toxicity control was 29.37 % already within 8 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item is considered to be readily biodegradable, since the pass level for the removal of 60 % ThCO2 in a 10-day window was fulfilled.

Executive summary:

The ready biodegradability of the test item was determined according to OECD TG 301B, EU method C.4-C and US EPA OPPTS 835.3110 under GLP. The test item as the nominal sole source of organic carbon was exposed to activated sludge originated from the aeration tank of a domestic waste water treatment plant in mineral medium and aerated by carbon dioxide-free air at a controlled rate in the dark. The biodegradation was followed over 28 days by determining the carbon dioxide produced. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the inoculum, only), abiotic and toxicity (containing both the test item and reference item) controls were examined. In this study all validity criteria regarding the inoculum, procedure and toxicity controls, inorganic carbon (IC) content of test item solution, and biodegradation values in the test item parallels were met. The percentage biodegradation of the test item reached a mean of 62.18 % (based on its theoretical carbon dioxide, ThCO₂) on the 17th day of the test. The pass level, 60 % of ThCO₂ was reached in about 9 - 10 days immediately following the attainment of 10 % biodegradation. The 10-day window was estimated from the evolved CO₂ determinations and the values were additionally derived from the biodegradation curve. The percentage biodegradation of the test item reached a mean of 70.19 % after 28 days. Based on the obtained values the test item is considered to be readily biodegradable. Based on its ThCO₂ the reference item Sodium benzoate was sufficiently degraded to 66.88 % already after 10 days, and to 78.87 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 43.95 % biodegradation was noted within 14 days and 53.06 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred already within 8 days). Because of the obtained microbial growth in the abiotic control, this group was excluded from the later evaluation, and correction of test item biodegradation value with abiotic processes was considered as not possible. In conclusion, the test item is considered to be readily biodegradable, since it fulfilled the pass level for ready biodegradability which is the removal of 60 % ThCO₂ in a 10-day window.

Description of key information

The test item is considered to be readily biodegradable, since the pass level for the removal of 60 % ThCO₂ in a 10-day window was fulfilled.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was determined according to OECD TG 301B, EU method C.4-C and US EPA OPPTS 835.3110 under GLP. The test item as the nominal sole source of organic carbon was exposed to activated sludge originated from the aeration tank of a domestic waste water treatment plant in mineral medium and aerated by carbon dioxide-free air at a controlled rate in the dark. The biodegradation was followed over 28 days by determining the carbon dioxide produced. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the inoculum, only), abiotic and toxicity (containing both the test item and reference item) controls were examined. In this study all validity criteria regarding the inoculum, procedure and toxicity controls, inorganic carbon (IC) content of test item solution, and biodegradation values in the test item parallels were met. The percentage biodegradation of the test item reached a mean of 62.18 % (based on its theoretical carbon dioxide, ThCO₂) on the 17th day of the test. The pass level, 60 % of ThCO₂ was reached in about 9 - 10 days immediately following the attainment of 10 % biodegradation. The 10-day window was estimated from the evolved CO₂ determinations and the values were additionally derived from the biodegradation curve. The percentage biodegradation of the test item reached a mean of 70.19 % after 28 days. Based on the obtained values the test item is considered to be readily biodegradable. Based on its ThCO₂ the reference item Sodium benzoate was sufficiently degraded to 66.88 % already after 10 days, and to 78.87 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 43.95 % biodegradation was noted within 14 days and 53.06 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred already within 8 days). Because of the obtained microbial growth in the abiotic control, this group was excluded from the later evaluation, and correction of test item biodegradation value with abiotic processes was considered as not possible. In conclusion, the test item is considered to be readily biodegradable, since it fulfilled the pass level for ready biodegradability which is the removal of 60 % ThCO₂ in a 10-day window.