2-(cyclohexylamino)ethanesulphonic acid

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:

2016-10-20 to 2017-08-15

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:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 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: activated sludge from a domestic waste water treatment plant

- Origin: sewage plant Balonfüred, Hungary

- Preparation of inoculum for exposure: The sludge was washed twice by centrifugation. The solid material was re-suspended in isotonic saline solution by shaking and centrifuging. This procedure was repeated twice. An aliquot of the final sludge suspension was weighted, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material/L was mixed with mineral medium and then aerated under test conditions (for 6 days) until use. The pH adjustment of activated sludge inoculum was not performed.


- Pretreatment: Pre-conditioning consisted of aerating (2 L/min) activated sludge (in mineral medium) for 6 days at the test temperature (20.1 - 21.9 °C).

- Concentration of sludge: 5 g/L

- Initial cell/biomass concentration: cell count of approx. 10e7 - 10e8 /L

- Water filtered: yes

- Type of filter used: cotton and coarse filter paper

Duration of test (contact time):

28 d

Initial conc.:

32 mg/L

Based on:

test mat.

Remarks:

corresponds to 15 mg organic DOC/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: According to the OECD TG 301B.
- Test temperature: 20.1 - 21.9 °C
- pH: 7.73 – 7.87 (measured at the start of test); 7.76 – 7.89 (measured at the end of test)
- pH adjusted: at study start to 7.38 (with 1N HCl)
- Aeration of dilution water: no
- Suspended solids concentration: approx. 25 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: glass flasks (5000 mL)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with CO2-free air at a rate of about 60 mL/min.
- Measuring equipment: The total carbon, inorganic carbon and dissolved organic carbon of the centrifuged samples were measured with a Total Organic Carbon Analyzer.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics: barium hydroxide adsorption bottles

SAMPLING
- Sampling frequency: on days 0, 2, 4, 6, 8, 11, 15, 20, 25, 28 and 29

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

Reference substance:

benzoic acid, sodium salt

Test performance:

Measurement of the evolved CO2 in all test groups (test item, inoculum control, procedure control and toxicity control) was performed every two to three days during the first eleven days of the test; thereafter every three to five days until test day 28 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 disconnect 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). The remaining absorption bottles were placed one position closer to the test flask, and a new absorption bottle containing 100 mL fresh 0.0125 M Ba(OH)2 was attached at the far end of the series.
Due to the intensive CO2 evolution in the 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, 100 mL fresh 0.0125 M Ba(OH)2 containing absorption bottles.
On test day 28 samples (about 50 mL from the test item containing flasks and inoculum control) were taken from the test flask for DOC analysis; 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. The titrations were performed.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

11.69

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: Based on ThCO2

Details on results:

Under the test conditions used, the percentage biodegradation of the test item reached a mean of 11.69 % after 28 days based on its theoretical carbon dioxide (ThCO2) which is less than the pass level for ready biodegradability of 60 %. The highest biodegradation value of 12.77 % was noticed on the 25th day of the test. The slight changes in biodegradation values reflect the biological variability of the test.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to 61.74 % already after 6 days, and to 76.22 % after 28 days of incubation, based on its ThCO2, thus confirming the suitability of the used activated sludge inoculum.

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 (0.59 %) was lower than 5 % of the TC.

- The total CO₂ evolution in the inoculum blank at the end of the test (on average 5.66 mg, corresponding to 1.89 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 at the biodegradation plateau the highest degradation difference of two replicates (~7 %) was calculated on the 15th day.

- The percentage degradation of the reference item reached the level of ready biodegradability (> 60 %) by exposure day 6. The percentage degradation of the reference item was 72.46 % on day 15.

- 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 33.36 % within 8 days.

 

Table 1: The Volume of Titrated 0.05 M HCl for Each Absorption Bottle

Test group	Flask number	The 0.05 M HCl consumption after n days of exposure (mL)
		2	4	6	8	11	15	20	25	28	28	29
Test item group	1	1.) 44.71	2.) 44.12	3.) 40.12	4.) 37.26	5.) 32.77	6.) 29.28	7.) 27.23	8.) 26.53	9.) 26.04	10.) 44.97	11.) 45.13
	2	1.) 45.02	2.) 45.23	3.) 39.67	4.) 38.12	5.) 31.94	6.) 28.13	7.) 26.46	8.) 25.87	9.) 26.31	10.) 46.16	11.) 44.27
	Mean	44.87	44.68	39.90	37.69	32.36	28.71	26.85	26.20	26.18	45.57	44.70
Inoculum control	3	1.) 46.40	2.) 46.33	3.) 46.17	4.) 46.12	5.) 45.41	6.) 43.96	7.) 43.87	8.) 45.05	9.) 44.25	10.) 46.29	11.) 42.13
	4	1.) 46.68	2.) 46.94	3.) 46.89	4.) 45.87	5.) 45.56	6.) 44.32	7.) 44.01	8.) 45.23	9.) 44.87	10.) 47.01	11.) 43.02
	Mean	46.54	46.64	46.53	46.00	45.49	44.14	43.94	45.14	44.56	46.65	42.58
Procedure control	5	1.) 34.26	4.) 41.56	7.) 27.46	10.) 23.63	13.) 21.32	16.) 18.11	19.) 17.52	22.) 16.34	23.) 2.95	24.) 10.13	25.) 3.12
			3.) 25.32	6.) 15.52	9.) 11.52	12.) 10.89	15.) 11.02	18.) 9.87	21.) 10.69
			2.) 15.99	5.) 8.13	8.) 4.23	11.) 3.52	14.) 2.95	17.) 3.05	20.) 4.23
Toxicity control	6	1.) 42.19		6.) 44.23	9.) 23.03	12.) 22.11	15.) 17.99	18.) 16.69	21.) 17.24	22.) 3.02	23.) 8.15	24.) 7.14
			3.) 34.26	5.) 20.19	8.) 11.95	11.) 11.23	14.) 10.31	17.) 9.91	20.) 10.55
			2.) 15.12	4.) 7.11	7.) 6.00	10.) 3.14	13.) 2.18	16.) 2.96	19.) 3.42

 

Table 2: The Calculated Weight of Produced CO₂

Test group	Flask number	The Calculated Weight of Evolved CO2 after n days of exposure (mg)
		2	4	6	8	11	15	20	25	28	28	29
Test item group	1	1.) 1.46	2.) 2.11	3.) 6.51	4.) 11.89	5.) 16.67	6.) 20.09	7.) 24.11	8.) 23.69	9.) 25.65	10.) 4.76	11.) 2.85
	2	1.) 1.12	2.) 0.89	3.) 7.01	4.) 10.95	5.) 17.58	6.) 21.35	7.) 24.96	8.) 24.42	9.) 25.36	10.) 3.45	11.) 3.80
	Mean	1.29	1.50	6.76	11.42	17.12	20.72	24.54	24.06	25.50	4.11	3.32
Inoculum control	3	1.) -0.40	2.) -0.32	3.) -0.14	4.) 2.15	5.) 2.76	6.) 3.94	7.) 5.81	8.) 3.32	9.) 5.62	10.) 3.31	11.) 6.15
	4	1.) -0.70	2.) -0.99	3.) -0.94	4.) 2.42	5.) 2.60	6.) 3.54	7.) 5.65	8.) 3.12	9.) 4.94	10.) 2.52	11.) 5.17
	Mean	-0.55	-0.65	-0.54	2.28	2.68	3.74	5.73	3.22	5.28	2.92	5.66
Procedure control	5	1.) 12.96	4.) 7.16	7.) 22.51	10.) 26.30	13.) 30.61	16.) 32.96	19.) 35.02	22.) 36.26
			3.) 22.79	6.) 35.64	9.) 39.62	12.) 42.09	15.) 40.76	18.) 43.44	21.) 42.47
			2.) 33.06	5.) 43.77	8.) 47.64	11.) 50.19	14.) 49.63	17.) 50.94	20.) 49.58	23.) 49.25	24.) 41.35	25.) 49.06
Toxicity control	6	1.) 4.24		6.) 4.06	9.) 26.96	12.) 29.74	15.) 33.09	18.) 35.94	21.) 35.27
			3.) 12.96	5.) 30.50	8.) 39.15	11.) 41.71	14.) 41.54	17.) 43.40	20.) 42.63
			2.) 34.01	4.) 45.06	7.) 45.69	10.) 50.61	13.) 50.48	16.) 51.04	19.) 50.47	22.) 49.17	23.) 43.53	24.) 44.64

The serial numbers of 1.), 2.), 3.) ….25.) remark 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.

For the accurate calculations the theoretical 50 mL value was substituted with the actual titrated values.

The titrated 0.05 M HCl volume is given for each absorption bottles in the Table 1.

 

Table 3: The Calculated Weight of Produced CO₂ corrected with Inoculum Values

The Calculated, Corrected Weight of Evolved CO2 after n days of exposure (mg)
Test group	Flask number	2	4	6	8	11	15	20	25	28
Test item group	1	2.01	2.77	7.05	9.61	13.99	16.35	18.38	20.47	19.41
	2	1.67	1.55	7.55	8.66	14.90	17.61	19.23	21.20	18.75
	Mean	1.84	2.16	7.30	9.14	14.44	16.98	18.80	20.83	19.08
Procedure control	5	12.96	63.01	101.92	111.28	120.21	119.60	123.67	125.08	125.80
Toxicity control	6	4.79	47.62	80.16	109.52	119.39	121.36	124.64	125.14	123.48

Remark: The negative values of inoculum control (calculated on the 2nd, 4th and 6th day of the test: for absorption bottles of 1, 2, 3) were taken into consideration as zero values (see Table 2).

At 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.

 

Table 4: 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	4	6	8	11	15	20	25	28
Test item group	1	1.23	1.70	4.32	5.89	8.57	10.02	11.26	12.54	11.89
	2	1.02	0.95	4.62	5.31	9.13	10.79	11.78	12.99	11.49
	Mean	1.13	1.32	4.47	5.60	8.85	10.40	11.52	12.77	11.69
Procedure control	5	7.85	38.17	61.74	67.42	72.83	72.46	74.93	75.78	76.22
Toxicity control	6	1.46	14.51	24.42	33.36	36.37	36.97	37.97	38.12	37.62

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

                                      mg CO₂ produced

% degradation = ------------------------------------------------ x 100

                            ThCO₂ x mg test substance added

Where the produced amount of CO₂ is originated from the Table 3, the ThCO₂ of the test item is 1.70 mg CO₂ / mg test item, and the test flask (with 3L reaction mixture) contained 3 x 32 mg test item.

 

Table 5: 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	4	6	8	11	15	20	25	28
Test item group	1	1.22	1.68	4.27	5.82	8.47	9.90	11.13	12.40	11.75
	2	1.01	0.94	4.57	5.25	9.02	10.66	11.64	12.83	11.35
	Mean	1.12	1.31	4.42	5.53	8.75	10.28	11.39	12.62	11.55
Toxicity control	6	2.62	13.82	22.28	27.34	31.91	31.22	34.80	34.74	35.64

 

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

                                    mg CO₂ produced

% degradation   = ---------------------------------------- x 100

                          mg TOC added in the test x 3.67

where the produced amount of CO₂ is originated from the Table 3, the TOC (that was considered to be equal with DOC) of the test item is 15 mg/L, and the test flask contained 3 L reaction mixture.

Analytical Results

Table 6: The Results of TC, IC Measurements, DOC Calculations   

Test item stock solution:				TC:		154.0 mg/L
				IC:		0.9069 mg/L
				DOC:		153.1 mg/L
Reference item stock solution:				TC:		153.8 mg/L
				IC:		1.015 mg/L
				DOC:		152.8 mg/L
TC, IC measurements, DOC calculations of the test mixtures
Test Groups	Flask Numbers	At the start of the test					At the end of the test
		TC (mg/L)	IC (mg/L)		DOC (mg/L)		TC (mg/L)	IC (mg/L)	DOC (mg/L)
Test Item	1	16.41	0.9662		15.44		14.36	0.5093	13.85
	2	16.18	0.9602		15.22		14.25	0.5332	13.72
Inoculum control	3	1.500	0.9299		0.5697		0.7441	0.4514	0.2927
	4	1.559	0.9587		0.6007		0.7209	0.3691	0.3518
Procedure control	5	16.41	1.022		15.39		#	#	#
Toxicity control	6	31.31	0.9827		30.33		15.85	1.198	14.65

#: Samples were not taken from the reference item containing procedure control flask at the end of the test.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was considered to be not readily biodegradable failing the pass level for ready biodegradability of 60 % of ThCO2 and 10-day window could not be determined. According to the test guidelines the pass level for ready biodegradability is 60 % of ThCO2.

Executive summary:

The ready biodegradability of the test item was determined in a study according to OECD TG 301B 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 (at a concentration of 25.71 mg/L that corresponded to 15 mg dissolved organic carbon/L (DOC/L)) 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) and toxicity (containing both the test item and reference item) controls were examined. The chosen test item concentration of 32 mg/L (that corresponded to 15 mg DOC/L) investigated in the main test was based on the results of the preliminary solubility test, based on the information about the toxicity of the test item, and based on the preliminary DOC and total organic carbon (TOC) determinations. 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. Under the test conditions, the percentage biodegradation of the test item reached a mean of 11.69 % after 28 days based on its theoretical carbon dioxide (ThCO₂) which is less than the pass level for ready biodegradability of 60 %. The highest biodegradation value of 12.77 % was noticed on the 25th day of the test. The slight changes in biodegradation values reflect the biological variability of the test. The total carbon (TC), IC and DOC contents of the samples taken from the test and reference item stock solutions and from the inoculated procedure control were determined at the start of the test. The TC, IC and DOC contents of the samples taken from the inoculated test item solutions in mineral medium, from the inoculum blank controls, and from the inoculated toxicity control were determined at the start and end of the test. The reference item Sodium benzoate was sufficiently degraded to 61.74 % already after 6 days, and to 76.22 % after 28 days of incubation based on its ThCO₂, 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 33.36 % biodegradation was noted within 8 days and 37.62 % 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). In conclusion, the test item was considered to be not readily biodegradable failing the pass level for ready biodegradability of 60 % of ThCO₂ and 10-day window could not be determined. According to the test guidelines the pass level for ready biodegradability is 60 % of ThCO₂.

Description of key information

The test item was considered to be not readily biodegradable (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was determined in a study according to OECD TG 301B 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 (at a concentration of 25.71 mg/L that corresponded to 15 mg dissolved organic carbon/L (DOC/L)) 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) and toxicity (containing both the test item and reference item) controls were examined. The chosen test item concentration of 32 mg/L (that corresponded to 15 mg DOC/L) investigated in the main test was based on the results of the preliminary solubility test, based on the information about the toxicity of the test item, and based on the preliminary DOC and total organic carbon (TOC) determinations. 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. Under the test conditions, the percentage biodegradation of the test item reached a mean of 11.69 % after 28 days based on its theoretical carbon dioxide (ThCO₂) which is less than the pass level for ready biodegradability of 60 %. The highest biodegradation value of 12.77 % was noticed on the 25th day of the test. The slight changes in biodegradation values reflect the biological variability of the test. The total carbon (TC), IC and DOC contents of the samples taken from the test and reference item stock solutions and from the inoculated procedure control were determined at the start of the test. The TC, IC and DOC contents of the samples taken from the inoculated test item solutions in mineral medium, from the inoculum blank controls, and from the inoculated toxicity control were determined at the start and end of the test. The reference item Sodium benzoate was sufficiently degraded to 61.74 % already after 6 days, and to 76.22 % after 28 days of incubation based on its ThCO₂, 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 33.36 % biodegradation was noted within 8 days and 37.62 % 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). In conclusion, the test item was considered to be not readily biodegradable failing the pass level for ready biodegradability of 60 % of ThCO₂ and 10-day window could not be determined. According to the test guidelines the pass level for ready biodegradability is 60 % of ThCO₂.