Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

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 June - 13 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dark
- Stability under test conditions: Asumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Insoluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Applied as supplied. Liquid dispensed on to polyethylene foil which was subsequently added to treatment vessels/ medium.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid dispensed on to polyethylene foil which was subsequently added to treatment vessels/ medium.

OTHER SPECIFICS: n/a

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600 000 inhabitant equivalents. Sampling date of activated sludge was on 26 June 2017.
- Laboratory culture: No
- Method of cultivation: n/a
- Storage conditions: Not reported
- Storage length: Not reported
- Preparation of inoculum for exposure: The dry solid content of the activated sludge was 3.5 g/L. It was determined by weight measurements after drying at 105 °C for 4 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
- Pretreatment: No
- Concentration of sludge: 3.5 g/L
- Initial cell/biomass concentration: n/a
- Water filtered: no
- Type and size of filter used, if any: n/a

Duration of test (contact time):

29 d

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: Per 1 L of demineralised water: 10 mL of an aquesous solution containing KH2PO4 (8.5 g/L), K2HPO4 (21.75 g/L), Na2HPO4.2H2O (33.40 g/L), NH4Cl (0.50 g/L); 1 mL CaCl2.2H2O (36.4 g/L); 1 mL MgSO4.7H2O (22.5 g/L) and 1 mL FeCl3.6H2O (0.25 g/L)
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 22 ± 2 ºC
- pH: not measured
- pH adjusted: no
- CEC (meq/100 g): not measured
- Aeration of dilution water: yes
- Suspended solids concentration: 3.5 g/L
- Continuous darkness: yes/
- Other: no

TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septa were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by magnetic stirrers with 2 cm stir bars. The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH.
- Number of culture flasks/concentration: 3 (with exception of toxic control where only 1 vessel was prepared).
- Method used to create aerobic conditions: The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.05 M NaOH (sodium hydroxide). At the end of the system is one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes.
- Method used to create anaerobic conditions: n/a
- Measuring equipment: IC measurement was performed with a total carbon analyser (TOC-5050A Shimadzu with an autosampler ASI-5000A) by purging the inorganic carbon with H3PO4 (25%) using a non-dispersive infrared (NDIR) detector.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH.
- Other: no

SAMPLING
- Sampling frequency: Day 0, 4, 7, 10, 14, 21, 28 and 29 (after addition of HCl).
- Sampling method: At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2-absorption flasks was determined as 2.75 mg/L. The IC in the reactors at the beginning of the test was 0.74 mg/L. On the 3rd, 7th, 10th, 14th, 21st and 28th day 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.
- Sterility check if applicable: no
- Sample storage before analysis: not reported
- Other: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: Reference control containing sodium benzoate in vessels corresponding to a concentration of 20.0 mg/L organic carbon.

STATISTICAL METHODS:

The theoretical CO2 amount of the test item (ThCO2) is calculated as follows;
ThCO2 [mg] = weight of added test item [mg] * carbon-content [mg/mg] * 44/12

The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber flasks as follows:
CO2 [mg/1500 mL] = IC [mg/L]* Volume absorber flask [L] *44/12

Hereby the volume of the absorber flask at the beginning of the test was 0.2 litre (200 mL) and the volume was reduced through each sampling by 4 mL. The factor 44/12 corresponds the molar weight-ratio of CO2 and C.

The amount of CO2 removed for IC-measurement is considered by adding up the CO2 content of each sampling to the current CO2 content of each absorber flask:
CO2 total (i, x) = CO2 absorber flask (i, x) + sum of CO2 sampling (i, x-1)
where;
CO2 total (i, x) = Total amount of CO2 [mg] absorbed in the ith absorber flask at the xth sampling including the amount removed by sampling
CO2 absorber flask (i, x) = CO2 [mg] absorbed in the ith absorber flask derived from IC-measurement at the xth sampling.
sum of CO2 sampling (i, x-1) = Sum of CO2 [mg] removed from the ith absorber flask with the 1th to x-1thsampling (sampling volume 4 mL each CO2 removed = IC [mg/L] * 0.004 [L]*44/12]).

The percentage biodegradation is calculated from:
BiodegradationCO2 [%] = 100*(CO2 Test reactor [mg] - CO2 Blank reactor [mg])/ThCO2 [mg]
where;
CO2 test reactor= Total CO2 evolution in the test reactor [mg]
CO2 Blank reactor = Total CO2 evolution in the blank reactor [mg] (mean of two vessels)

Reference substance:

other: sodium benzoate

Test performance:

The reference compound sodium benzoate reached the pass levels for ready biodegradability within 7 days indicating the reliability of the test system.

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Details on results:

No degradation could be observed within the test duration (28 days, after acidification). The biodegradation extent of the test item at the end of the test showed a negative value (-30.1%).

The negative degradation value during the test may indicate a slight inhibitory effect on the inoculum.

Table 1       Biodegradation (%)

Day	% Biodegradation
	Procedural Control	Test Item			Reference Control			Toxicity Control
	Mean	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3
0	0	0	0	0	0	0	0	0
4	9.3	-8.2	-4.4	-3.6	52.4	52.2	48.7	31.5
7	17.5	-16.5	-12.5	-13.8	70.4	65.0	61.9	35.0
11	23.7	-16.4	-13.1	-17.9	82.4	79.8	72.1	42.8
14	30.2	-22.4	-21.4	-27.3	81.2	76.8	76.8	42.3
21	38.2	-28.2	-21.8	-34.5	79.7	77.1	75.7	40.6
28	42.9	-32.2	-17.4	-34.4	79.5	74.8	80.3	41.9
29	46.2	-35.6	-20.2	-34.6	74.0	69.7	75.6	38.9

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

No degradation could be observed within the test duration (28 days, after acidification). The biodegradation extent of the test item at the end of the test showed a negative value (mean: -30.1 %).

The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).

The negative degradation value during the test may indicate a slight inhibitory effect on the inoculum.

Executive summary:

OECD 301B (2017) - The biodegradability of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) was assessed in an OECD 301B closed bottle test. The test substance was exposed to a relatively low number of microorganisms present in mineral medium inoculated with activated sewage sludge, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

a) An inoculated control, in triplicate, consisting of inoculated mineral medium.

b) The procedure control containing the reference item (sodium benzoate), in triplicate, in inoculated mineral medium to give a final concentration of 20 mg carbon/L.

c) The test item, in triplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.

d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 30 mg carbon/L to act as a toxicity control (one vessel only)

Evolved CO₂was captured in 0.2 M sodium hydroxide traps from which 4 mL aliquots were removed periodically (Day 0, 3, 7, 10, 14, 21, 28 and 29) throughout the test for inorganic carbon (IC) analysis. The samples were analyzed for IC using either a Shimadzu TOC analyser with each analysis was carried out in at least triplicate.

Calculated results concluded that the test item did not biodegrade after 28 days (mean biodegradation = -30.1 %, n=3)

Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable.

Description of key information

Under test conditions no biodegradation observed; OECD 301B; Brunswik-Titze, A. (2017)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

OECD 301B (2017) - The biodegradability of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) was assessed in an OECD 301B closed bottle test. The test substance was exposed to a relatively low number of microorganisms present in mineral medium inoculated with activated sewage sludge, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

a) An inoculated control, in triplicate, consisting of inoculated mineral medium.

b) The procedure control containing the reference item (sodium benzoate), in triplicate, in inoculated mineral medium to give a final concentration of 20 mg carbon/L.

c) The test item, in triplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.

d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 30 mg carbon/L to act as a toxicity control (one vessel only)

Evolved CO₂was captured in 0.2 M sodium hydroxide traps from which 4 mL aliquots were removed periodically (Day 0, 3, 7, 10, 14, 21, 28 and 29) throughout the test for inorganic carbon (IC) analysis.The samples were analyzed for IC using either a Shimadzu TOC analyser with each analysis was carried out in at least triplicate.

Calculated results concluded that the test item did not biodegrade after 28 days (mean biodegradation = -30.1 %, n=3)

Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable.