4-(cyclohexylamino)butane-1-sulfonic 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:

23 Apr 2019 - 23 May 2019

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 July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Correction factor for purity: not required
- Stability at higher temperatures: stable
- Solubility in water: 110 g/L

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 3.2 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9.6 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

12 mg/L

Based on:

TOC

Initial conc.:

23.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: According to OECD 301B
- Test temperature: 22-23 °C
- pH: at start: 7.6; t = 14 d: 7.8 (procedural and toxicity control); t = 28 d: 7.6 (blank controls and test solutions)
- pH adjusted: yes, one blank control and the procedural control from 7.7 to 7.6 using 1 M HCl adjusted at the start of the test
- Continuous darkness: yes
- Continuous aeration and stirring: yes
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for procedural control, 1 for toxicity control.
- Method used to create aerobic conditions: aeration with synthetic air (mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), CO2 < 1 ppm) at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

CONTROLS
- Inoculum blank: containing only inoculum
- Procedural control: containing reference item (sodium acetate) and inoculum
- Toxicity control: containing test item, reference item (sodium acetate) and inoculum

PREPARATION OF TEST SOLUTIONS
Since the test item was easily soluble in water, test media were prepared using a stock solution of 1 g/L in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). A weighed amount of 499.55 mg of test item was dissolved in Milli-RO water and made up to 500 mL. After stirring for 60 minutes, the final stock solution was clear and colourless (pH 5.8). Aliquots of 47 mL of the stock solution were added to test item bottles A and B and the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres). Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms.

SAMPLING
- Sampling frequency: Titrations were made on day 2, 5, 8, 12, 15, 19, 23 and 29 for the inoculum blank and test item. Titrations for the procedural and toxicity control were made on day 2, 5, 8, 12 and 15.
- Sampling method: titration of Ba(OH)2 in the gas scrubbing bottles.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

29 d

Remarks on result:

other: HCl added on the 28th day (last CO2-measurement on the 29th day).

Details on results:

- ThCO2 of the test item was calculated to be 1.87 mg CO2/mg and ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg
- Biodegradation test item: Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of test item (1% and 0%, based on ThCO2). See Table in 'Any other information on results incl. tables' for details.
- Toxicity control: More than 25% biodegradation occurred within 14 days (41%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- The temperature and pH during the exposure period were within the prescribed range of the study plan.
- See attached illustration for biodegradation curves of Procedural Control, Toxicity Control and Test Bottles.

Results with reference substance:

The reference substance showed a normal biodegradation curve and reached > 60% (92%) biodegradation within 14 days.

Day	Cumulative CO2 production in Blank Bottles (mg)	Cumulative CO2 production in Procedural Control (mg)	Biodegradation in Procedural Control (%)	Cumulative CO2 production in Toxicity Control (mg)	Biodegradation in Toxicity Control (%)	Cumulative CO2 production in Test Bottle A (mg)	Biodegradation in Test Bottle A (%)	Cumulative CO2 production in Test Bottle B (mg)	Biodegradation in Test Bottle B (%)
2	4.1	19.0	22	20.2	12	0.0	0	0.0	0
5	10.4	42.4	50	42.9	25	0.0	0	0.0	0
8	18.1	58.7	69	57.7	33	0.0	0	0.0	0
12	26.6	69.4	81	65.3	38	0.5	1	0.0	0
15	31.6	78.7	92	70.3	41	0.8	1	0.0	0
19	39.2					0.8	1	0.0	0
23	46.0					0.8	1	0.0	0
29	55.0					0.8	1	0.0	0
29	59.3					0.8	1	0.0	0
29	62.4					0.8	1	0.0	0

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The substance was not readily biodegradable in the CO2 Evolution Test, performed according to OECD guideline 301 B and GLP principles.

Executive summary:

In a CO2 Evolution Test performed according to OECD guideline 301B and GLP principles, the substance was evaluated for its ready biodegradability. The test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days. The substance was tested in duplicate at a target concentration of 23.5 mg/L, corresponding to 12 mg TOC/L. Two inoculum blanks, one procedural control (sodium acetate) and one toxicity control (substance plus sodium acetate) were included. Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item. Since 41% biodegradation occurred in the toxicity control within 14 days, the substance was assumed not to inhibit microbial activity. All acceptability criteria for the test were met and the study is therefore considered to be valid. Based on the obtained results in this test, the substance is concluded to be not readily biodegradable.

Description of key information

The substance was not readily biodegradable in the CO2 Evolution Test, performed according to OECD guideline 301 B and GLP principles.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

In a CO2 Evolution Test performed according to OECD guideline 301B and GLP principles, the substance was evaluated for its ready biodegradability. The test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days. The substance was tested in duplicate at a target concentration of 23.5 mg/L, corresponding to 12 mg TOC/L. Two inoculum blanks, one procedural control (sodium acetate) and one toxicity control (substance plus sodium acetate) were included. Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item. Since 41% biodegradation occurred in the toxicity control within 14 days, the substance was assumed not to inhibit microbial activity. All acceptability criteria for the test were met and the study is therefore considered to be valid. Based on the obtained results in this test, the substance is concluded to be not readily biodegradable.