Sodium 1,4-dicyclohexyl sulphonatosuccinate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Remarks:

Enhancement (prolongation of standard 301B test to 60 days)

Type of information:

experimental study

Adequacy of study:

key study

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:

July 1992
Enhancement (prolongation of standard 301B test to 60 days)

Deviations:

no

Principles of method if other than guideline:

Enhancement (prolongation of standard 301B test to 60 days)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant AZV Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600,000 inhabitant equivalents. Sampling date of activated sludge was on 23 November 2020. The dry solid content of the activated sludge was 3.3 g/L. It was determined by weight measurements after drying at 105°C for 4.25 hours (mean of triplicate measurements). Before use the sludge was washed twice with tap-water by settling and decanting the supernatant

Duration of test (contact time):

60 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
A: Potassium dihydrogenphosphate KH2PO4 8.50 g
Dipotassium hydrogenphosphate K2HPO4 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
Ammonium chloride NH4Cl 0.50 g
are dissolved in demineralised water and made up to 1 litre. The pH of the solution should be 7.4.
B: Calcium chloride dihydrate CaCl2 * 2H2O 36.4 g
is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g
is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.
For preparation of both of the mineral media 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre. pH was measured and adjusted if necessary to 7.4 +/- 0.2.

- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 20.5 – 22.5°C
- pH: 7.4 +/- 0.2
- pH adjusted: yes (if necessary)
- Suspended solids concentration: 30 mg/L dry solids
- Continuous darkness: yes


TEST SYSTEM
● Compressor NO10.AN 18, KNF Neuberger, Freiburg
● 1000 mL gas wash bottles with Teflon-sealing, Thoma, Freiburg
● Magnetic stirrer, ‘MONO direct’ with stir bars 2 cm, H+P Labortechnik AG, Oberschleißheim
● Row of air-tubes (air distributor) with two input and 22 output channels, Thoma, Freiburg
● Perforated plugs with PE-tubes (2.8/2.0 mm), Thoma, Freiburg
● 2000 mL gas wash flasks with GL14 hole-caps and frit pipes (reactors), Gerätebau Ochs, Bovenden-Lenglern
● Two 250 mL gas wash bottles connected in series with GL14 hole caps for each channel and frit pipes (CO2-absorber flask), Gerätebau Ochs, Bovenden-Lenglern
● Total carbon analyzers TOC-L with autosamplers, Shimadzu Deutschland, Duisburg
● Sealing Parafilm „M“, Pechiney Plastic Packaging, Chicago, USA
● Analytical balance BP 221S, Sartorius AG Göttingen
● Precision balance LP 6200S, Sartorius AG Göttingen
● Thermometer VWRI620-2042 with min/max-display, VWR International GmbH, Darmstadt
● Adjustable micropipettes 200 µL, 1 mL and 5 mL, Eppendorf, Wesseling/Berzdorf
● Syringe for sampling, Braun Injekt 5 mL, 2 mL, Melsungen
● Needle for sampling, Sterican Braun 21 G, Melsungen
● Drying oven, Memmert, Schwabach
● Ultrasonic device, Branson Sonifier, G. Heinemann Ultraschall- und Labortechnik, Schwäbisch Gmünd

SAMPLING
- Sampling frequency: 3rd, 7th, 10th, 14th, 17th, 21st, 28th, 35th, 42th, 49th, 56th and 60th
- Sampling method: 8 mL NaOH were sampled from the first of two CO2-absorber flasks connected in line and the IC's were determined.
- Sterility check if applicable: no


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no


STATISTICAL METHODS: none

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Preliminary tests were set up to check (1) general temperature development of a solution during treatment with ultrasonic and (2) homogeneity of the stock solution.

(1) General temperature development of stock solution during the treatment with ultrasonic
Material and method:
A general preliminary test on temperature development during ultrasonic treatment was carried out beforehand. A Branson Sonifier 450-D was used for this purpose. A stock solution with the test substance (10 g/L) in mineral medium was sonicated at a frequency of approximately 20,000 kHz and an amplitude of 80%. The temperature development during sonication was recorded.
Results:
Quite a strong temperature development started after only a few seconds, the temperature increased to >24°C within a few seconds.
Conclusion:
In agreement with the sponsor we decided, that the temperature range of the stock solution should not exceed 24°C. Therefore, in the main test, the stock solution was cooled in a water bath during the ultrasonication and the temperature was documented. During sonication the temperature ranged from 19.3 to 24.0°C.
(2) Homogeneity of the stock solution
Material and method:
A stock solution with a test item concentration of 10 g/L was prepared with mineral medium. Therefore 1000 mg of the test item was diluted in 100 mL mineral medium (see photo 1). The test item dissolved immediately. The stock solution was not heated.
Results:
A visual assessment of the stock solution showed a homogenous, clear liquid after a stirring time of 1 minute at room temperature.
Conclusion:
In order to obtain a homogeneous stock solution in the main test, the stock solution was prepared with a stirring time of 1 – 2 minutes at room temperature.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

48.7

St. dev.:

8

Sampling time:

28 d

Remarks on result:

other: without ultra sonication of stock solution prior to application

Key result

Parameter:

% degradation (CO2 evolution)

Value:

27.1

St. dev.:

2.3

Sampling time:

28 d

Remarks on result:

other: with ultra sonication of stock solution prior to application

Parameter:

% degradation (CO2 evolution)

Value:

76.9

St. dev.:

5.5

Sampling time:

60 d

Remarks on result:

other: without ultra sonication of stock solution prior to application

Key result

Parameter:

% degradation (CO2 evolution)

Value:

43.8

St. dev.:

12.9

Sampling time:

60 d

Remarks on result:

other: with ultra sonication of stock solution prior to application.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Ultimate biodegradation under enhanced OECD 301 B conditions exceeded the trigger value of 60% on day 60. Hence the substance is considered not persistent in the environment.

Conclusions:

The study results show that the substance is ultimately biodegraded by more than 60% in 60 days. Replicate values were 74%, 74% and 83% (average = 77%)

Executive summary:

Stock solution without ultrasonic treatment:

The mean degradation of the test item after 28 days was 48.7% ± 8.0% (mean of three replicates, see table 1). The difference between the replicates was 15.9%, the criterion of 20% difference of extremes of replicate values is therefore met. The test is valid according to OECD 301 B (July 1992). The test substance is not readily biodegradable.

 

For enhanced biodegradability testing, the test duration was prolonged to 60 days. On that day the degradation of the test item was 76.9% ± 5.5% (mean of three replicates, with considering the IC in the liquid phase, see table 1 and figure 1). The difference between the replicates was 9.7%.

 

Stock solution with ultrasonic treatment:

The mean degradation of the test item after 28 days was 27.1% ± 2.3% (mean of four replicates, see table 3). The difference between the replicates was 5.4%, the criterion of 20% difference of extremes of replicate values is therefore met. The test is valid according to OECD 301 B (July 1992). The test substance is not readily biodegradable.

 

For enhanced biodegradability testing, the test duration was prolonged to 60 days. On that day the degradation of the test item was 43.8% ± 12.9% (mean of four replicates, with considering the IC in the liquid phase, see table 3 and figure 2). After day 28, the difference between the replicates was generally < 20%, with the exception of day 60, it was 30%. However, three of the replicates were similar, with a difference between the replicates of 9.9%.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-29 to 2013-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed under GLP and according to appropriate methods to address this endpoint.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: non-adapted and adapted activated domestic sludge (test groups 1 and 2, respectively)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from municipal sewage STP D-31137 Hildesheim. The sludge of that STP comprises mostly municipal sewage and hardly any industrial chemical waste.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: Not applicable.
- Storage length: Not applicable.
- Preparation of inoculum for exposure: The activated sludge was washed twice with tap water. The dry sludge concentration was determined and an appropriate volume of inoculum was chosen to reach a dry sludge concentration of 0.2 - 1.0 g/L in the test vessels and a ratio of DOC of test compound / inoculum in the range of 1:2.5 - 1:4.
- Pretreatment: Side experiment with adapted sludge: On day 0, 1 L of activated sludge was spiked with 50 mg/L yeast extract. 4 mg C/L of the test item was added. The inoculum was covered and stirred continuously. On day 7 and 10 after preparation of the inoculum 8 mg C/L of the test item was added. Prior to application the dry sludge concentration was determined and an appropriate volume of inoculum was chosen to reach a dry sludge concentration of 0.2 - 1.0 g/L in the test vessels and a ratio of DOC of test compound / inoculum in the range of 1:2.5 - 1:4.
- Concentration of sludge: See below, biomass concentration.
- Initial cell/biomass concentration: Non-adapted inoculum: 6.86 g dry sludge/L. Adapted inoculum: 5.87 g dry sludge/L. Ratio of DOC of test compound to inoculum dry weight: 1:4.0 (non-adapted and adapted inoculum).
- Water filtered: See above.
- Type and size of filter used, if any: No data.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

51.8 mg/L

Based on:

other: Carbon

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 302 B.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20.3 to 23 °C.
- pH adjusted: The pH was adjusted in every test vessel to 6.5 - 8.0, if necessary.
- pH value during the test: 6.7 to 7.8 in all test vessels.
- CEC (meq/100 g): No data are reported.
- Aeration of dilution water: Aeration with CO2 free air under continuous stirring.
- Suspended solids concentration: Not determined, assumed: ambient conditions.
- Continuous darkness: No information, ambient conditions assumed.

TEST SYSTEM
- Culturing apparatus: Several bottles in series (reactor system): 2 L test vessels.
- Number of culture flasks/concentration:
Test group 1 (non-adapted sludge)
Reactors 1 and 2: Test concentration 100 mg/L test material (51.8 mg C/L).
Reactors 3 and 4: Blank control.
Reactor 5: Reference substance - Diethylene glycol 120 mg/L (54.4 mg C/L).
Reactor 6: Abiotic control - Test item in test concentration without inoculum, poisoned with
10 mL/L HgCl2 solution (10 g/L).
- Number of culture flasks/concentration:
Test group 2 (adapted activated sludge):
Reactors 1 and 2: Test concentration 100 mg/L test material (51.8 mg C/L).
Reactor 3: Blank control.
Reactors 4 and 5: Reference substance - Diethylene glycol 120 mg/L (54.4 mg C/L).
- Method used to create aerobic conditions: Continuous aeration with CO2 free air, mixing of the test solutions by magnetic stirring (continuously).
- Method used to create anaerobic conditions: NA.
- Measuring equipment: DOC Analyzer.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Yes, but aerated with CO2 free air.
- Details of trap for CO2 and volatile organics if used: Not applicable.

SAMPLING
- Sampling frequency:
Non adapted activated sludge: days 0 (3 hours), 2, 7, 14, 20, 28 after incubation initiation.
Adapted activated sludge: days 0 (3 hours), 3, 7, 14, 21, 28 after incubation initiation.
- Sampling method: Removal and filtration of aliquots.
- Sterility check if applicable: Not reported.

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two replicates.
- Abiotic sterile control: Yes (one replicate).
- Toxicity control: No.
- Reference substance: Yes (one replicate).
- Other:

STATISTICAL METHODS: NA

Reference substance:

benzoic acid, sodium salt

Test performance:

No events are reported, which might have affected the quality of the study.

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

14 d

Remarks on result:

other: Non-adapted sludge

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

14 d

Remarks on result:

other: Adapted sludge

Details on results:

The biodegradation for the replicates with non-adapted activated sludge reached the 10 % level (beginning of biodegradation) after 3 days. After 7 days the biodegradation came to 52 %. The pass level of 70 % was reached on day 10 and the biodegradation came to a maximum of 100 % after 14 days. For the replicates with adapted activated sludge, the start of the biodegradation (10% level) was reached after one day. After 7 days, a biodegradation of 78 % was determined and the pass level of 70 % was reached after 5 days. The maximum of degradation was 100 % after 14 days. No significant physico-chemical elimination occurred in the sterile control after 28 days (maximum 3%).

Results with reference substance:

Biodegradation of the reference substance reached 100 % within 14 days of incubation. Hence the test is rendered valid.

Interpretation of results:

other: Inherently ultimately biodegradable

Conclusions:

The substance is inherently ultimately biodegradable by non-adapted sludge. Biodegradation was reached within 3 days (10% level passed) and the pass level of 70% was reached on day 10. There was no elimination by sorption and abiotic degradation and hence elimination is due to mineralization of the test substance.

Executive summary:

In a valid GLP study according to OECD 302 B the test item was aerobically exposed at 100 mg/L test material (51.8 mg C/L) to standard OECD mineral medium and activated sludge obtained from a domestic STP. Two test groups were analyzed: Test group 1 used non-adapted sludge and test groups 2 used adapted sludge. The dry sludge concentration was 6.86 g/L (adapted sludge) and 5.87 g/L (non-adapted sludge), respectively. The ratio DOC to activated sludge dry weight was 1:4 at the beginning. For sludge adaptation, 50 mg/L yeast and 4 mg C/L (test material) were added to the inocculum which was then maintained under test conditions until start of the main test. On days 7 and 10 further 8 mg C/L test substance were added. The thus adapted sludge was used after 14 days to run test group 2. Exposure of both groups was for 28 days at ambient light conditions, under permanent stirring and between 20.3 and 23°C. Per test group, six test vessels with a volume of 2 L were stocked with 2 L volume, which was constantly aerated with CO2 free air and stirred permanently. Each reactor had received inocculum and mineral medium. Per test, the following replicates were incubated: Test item (100 mg C/L, two replicates), blank control (two replicates), reference substance (120 mg C/L of diethylene glycol, one replicate) and an abiotic control (sterilized with a 10 mL/L HgCl2 solution). DOC of filtered samples was analyzed by a DOC analyzer at 3 hours (non-adapted sludge only) and on days 2(3), 7, 14, 20(21) and 28 (number in brackets: adapted sludge). The 14-day results of the functional control showed that a biodegradation rate of ≥ 99 % was reached with non-adapted and adapted activated sludge. These result validates the testing procedure. The biodegradation for the replicates with non-adapted activated sludge reached the 10 % level (beginning of biodegradation) after 3 days. After 7 days the biodegradation came to 52 %. The pass level of 70 % was reached on day 10 and the biodegradation came to a maximum of 100 % after 14 days. For the replicates with adapted activated sludge, the start of the biodegradation (10% level) was reached after one day. After 7 days, a biodegradation of 78 % was determined and the pass level of 70 % was reached after 5 days. The maximum of degradation was 100 % after 14 days. No significant physico-chemical elimination occurred in the sterile control after 28 days (maximum 3%). Therefore it can be concluded, that the decrease of the test item is due to biodegradation. It can be concluded, that the substance is inherently ultimately biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Additional information

A study for ready biodegradability is available. The test was performed according to OECD 301 B and is valid according to the guideline criteria. After incubation for 28 days, 12% (18-5%) of the test material was ultimately degraded.

Conclusion: The substance is not readily biodegradable.

 

A study for inherent biodegradability is available. The test was performed according to OECD 302 B and is valid according to the guideline criteria. After incubation for 14 days, 99% of the test material was ultimately degraded. Conclusion: The substance is inherently biodegradable. Specific criteria were not fulfilled: On day 7 biodegradation reached 52%.

 

A key study for ready and enhanced biodegradability is available. It includes 2 different set-ups: (1) Stock solutions were not homogenized prior to use (2) Stock solution were homogenized by ultrasonic treatment prior to use. Rationale for two set-up 2: Minimizing variability between the replicates. The test was performed according to OECD 301 B and is valid according to the guideline criteria. Set-up 1: After incubation for 28 days, 49 ± 8% (40-50-56%) of the test material was ultimately degraded. After incubation for 60 days, 77 ± 6% (77-74-83%) of the test material was ultimately degraded. Set-up 2: After incubation for 28 days, 27 ± 2% (24-28-28-29%) of the test material was ultimately degraded. After incubation for 60 days, 44 ± 13% (32-39-42-62%) of the test material was ultimately degraded.

 

A further study for ready and enhanced biodegradability is available, supporting the results of the corresponding key study. The test was performed according to OECD 301 F in triplicate and is valid according to the guideline criteria. At the end of the 10-day window, 21 ± 8% (13-21-29) of the test material was ultimately degraded with a difference of extremes of 15%. After 28 days, 46 ± 23% (30-58-61) were ultimately degraded, however the difference between extremes was 42%. Hence day 28 values cannot be use for the interpretation of results on ready biodegradation. After incubation for 60 days, 62 ± 22% (38-68-80%) of the test material was ultimately degraded.

 

A repeat study for ready and enhanced biodegradability is available. It includes 2 different set-ups: (1) Stock solutions were not homogenized prior to use (2) Stock solution were homogenized by ultrasonic treatment prior to use. Rationale for two set-up 2: Minimizing variability between the replicates. The test was performed according to OECD 301 B with four replicates and is valid according to the guideline criteria. Set-up 1: After incubation for 28 days, 32 ± 5% (25-32-36-37%) of the test material was ultimately degraded. After incubation for 60 days, 50 ± 5% (43-48-51-56%) of the test material was ultimately degraded. Set-up 2 did not meet the validity criteria for a OECD 301 study, since the difference of extremes exceeded 20% on the 10 day window and on day 28.