Sodium 1,4-diisodecyl sulphonatosuccinate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready 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 day
- 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 18.9 to 24.0°C.

(2) Homogenicity 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 750 mg of the test item was diluted in 75 mL mineral medium (see photo 1). The stock solution was stirred with a stirring fish at room temperature until a homogeneous solution was obtained (see photo 2). The stock solution was not heated.
Results:
A visual assessment of the stock solution showed a homogenous, opaque liquid after a stirring time of 30 minutes at room temperature (see photo 3).
Conclusion:
In order to obtain a homogeneous stock solution in the main test, the stock solution was prepared with a stirring time of 30 minutes at room temperature.

Parameter:

% degradation (CO2 evolution)

Value:

14.4

St. dev.:

6.2

Sampling time:

28 d

Remarks on result:

other: without ultra sonication of stock solution prior to application

Parameter:

% degradation (CO2 evolution)

Value:

26.2

St. dev.:

3.7

Sampling time:

28 d

Remarks on result:

other: with ultra sonication of stock solution prior to application

Parameter:

% degradation (CO2 evolution)

Value:

63.9

St. dev.:

11.4

Sampling time:

60 d

Remarks on result:

other: without ultra sonication of stock solution prior to application

Key result

Parameter:

% degradation (CO2 evolution)

Value:

78.1

St. dev.:

14

Sampling time:

60 d

Remarks on result:

other: with ultra sonication of stock solution prior to application

Details on results:

Stock solution without ultrasonic treatment:

The mean degradation of the test item after 28 days was 14.4% ± 6.2% (mean of three replicates). The difference between the replicates was 15.1%, the criterion of 20% difference of extremes of replicate values is therefore met.

For enhanced biodegradability testing the test duration was prolonged up to 60 days. At the end of the test (60d), the degradation of the test item was 63.9% ± 11.4% (mean of three replicates, with considering the IC in the liquid phase). The difference between the replicates was 27.7%. But the criterion of 20% difference of extremes of replicate values is not applicable to the enhanced version of the test.



Stock solution with ultrasonic treatment:

The mean degradation of the test item after 28 days was 26.2%± 3.7% (mean of four replicates). The difference between the replicates was 10%, the criterion of 20% difference of extremes of replicate values is therefore met.

For enhanced biodegradability testing the test duration was prolonged up to 60 days. At the end of the test (60d), the degradation of the test item was 78.1% ± 14.0% (mean of four replicates, with considering the IC in the liquid phase). In the enhanced phase of the test the variation of the replicates slowly increased and on day 60 the difference of extremes was 26.3% and thus higher than 20%. But the criterion of 20% difference of extremes of replicate values is not applicable to the enhanced version of the test.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Ultimate biodegradation under enhanced OECD 301 F conditions exceeded 60% on day 60.

Conclusions:

The study results show that the substance was ultimately biodegraded by more than 60% in 60 days. Stock solutions ultrasonicated: After incubation for 60 days, 64 ± 11% (51-62-79%) of the test material was ultimately degraded; Stock solutions not ultrasonicated: After incubation for 60 days, 78 ± 14% (60-70-87-96%) of the test material was ultimately degraded.

Executive summary:

Stock solution without ultrasonic treatment:

The mean degradation of the test item after 28 days was 14.4% ± 6.2% (mean of three replicates). The difference between the replicates was 15.1%, the criterion of 20% difference of extremes of replicate values is therefore met.

For enhanced biodegradability testing the test duration was prolonged up to 60 days. At the end of the test (60d), the degradation of the test item was 63.9% ± 11.4% (mean of three replicates, with considering the IC in the liquid phase). The difference between the replicates was 27.7%. But the criterion of 20% difference of extremes of replicate values is not applicable to the enhanced version of the test.

 

Stock solution with ultrasonic treatment:

The mean degradation of the test item after 28 days was 26.2%± 3.7% (mean of four replicates). The difference between the replicates was 10%, the criterion of 20% difference of extremes of replicate values is therefore met.

For enhanced biodegradability testing the test duration was prolonged up to 60 days. At the end of the test (60d), the degradation of the test item was 78.1% ± 14.0% (mean of four replicates, with considering the IC in the liquid phase). In the enhanced phase of the test the variation of the replicates slowly increased and on day 60 the difference of extremes was 26.3% and thus higher than 20%. But the criterion of 20% difference of extremes of replicate values is not applicable to the enhanced version of the test.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Conduct of Study: 7 January 2016 (experimental starting date), 4 February 2016 (experimental completion date)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed under GLP and according to appropriate methods to address this endpoint. The TOC content of the test item was determined by a third laboratory under the quality management ISO/IEC 17025, and is thus excluded from the statement of GLP compliance. Due to technical problems with the validated TOC Analyzer, DOC measurements before and after sludge addition, after 3 hours and after 1 day were made on a technically equal but not yet validated TOC Analyzer. These measurements are, therefore, excluded from the statement of compliance. However, the precision of the results of the test are equal to the validated equipment. The procedure control (one replicate) for the mineralization part of the test (CO2 evolution measurements) did not yield reasonable results after days 7, most likely due to system leakage. Until then 51% of the reference substance had been mineralized. The S-shape of the duplicated mineralization curves however indicates, that the test item systems were operated without leakage and thus the mineralization results are considered valid.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

In addition to requirements of the guidelines (DOC determination), evolved CO2 was measured at several intervals during the study. Rationale: In order to investigate effects of adsorption and mineralization on elimination of the substance.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Non-adapted activated sludge from the aeration tank of the STP Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant, not adapted.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: The activated sludge was used after sampling from the treatment plant without adaptation. However, the sludge was pre-conditioned for 3 days (aerated but not fed).
- Storage length: 3 days.
- Preparation of inoculum for exposure: Prior to the test the sludge was washed twice with tap water and once with mineral medium.
- Pretreatment: None
- Concentration of sludge: 0.2 g/l dry matter in the final mixture.
- Initial cell/biomass concentration: No data.
- Water filtered: No data.
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Initial conc.:

113 mg/L

Based on:

test mat.

Initial conc.:

47.5 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation:

CO2 evolution

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: 22 ± 2 °C.
- pH adjusted: The pH-value was checked and adjusted to pH 7.4 +/- 0.2 with HCl. At the end of the test the pH values of the blank and procedure controls were 7.0 and 6.9, respectively. The pH values of the test units were 7.1 and 7.0.
- CEC (meq/100 g): No data are reported.
- Aeration of dilution water: Aeration with CO2 free air under continuous stirring.
- Suspended solids concentration: 0.2 g/L dry matter (sludge).
- Continuous darkness: Yes. The test was conducted in temperature controlled dark room.

TEST SYSTEM
- Culturing apparatus: 2.2 litre closed glass bottle containing a total volume of test solution of 2 l; aerated with CO2-free air and fitted to gas-absorption bottles containing 125 ml of 0.13 M KOH.
- Number of culture flasks/concentration:
Reactors 1 to 3 (Test Group T): Test concentration 113 mg/L (47.5 mg/L TOC).
Reactors 4 and 6 (Test Group B, Procedure Control): 110 mg/L (50 mg/L TOC).
Reactors 7 and 9: Blank control, containing test medium and activated sludge.
- 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 (elimination), TOC analyser (mineralization).
- 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: Gas-absorption bottles containing 125 ml of 0.13 M KOH.
SAMPLING
- Sampling frequency:
DOC: Prior and after sludge addition, 3 hours, 1,4,7,11,14,18,21,25,27 and 28 days.
CO2: After incubation initiation (Day 0), and on days 7, 14, 21 and 28.
- Sampling method: Removal and filtration of aliquots. Centifugation.
- Sterility check if applicable: NA.

- Other:

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

STATISTICAL METHODS: NA

Reference substance:

diethylene glycol

Test performance:

The procedure control (one replicate) for the mineralizsation part of the test (CO2 evolution measurements) did not yield reasonable results after days 7, most likely due to system leakage. Until then 51% of the reference substance had been mineralized. The S-shape of the duplicated mineralization curves however indicate that the test item systems were operated without leakage and thus the mineralization results are considered valid.

Parameter:

% degradation (DOC removal)

Value:

91

Sampling time:

28 d

Remarks on result:

other: Non-adapted sludge

Parameter:

% degradation (inorg. C analysis)

Value:

58

Sampling time:

14 d

Remarks on result:

other: Non-adapted sludge

Details on results:

Based on DOC measurements, Sulfosuccinic (isoFA C10)diE, Na was eliminated by more than 70% at the end of the incubation period, i.e. average 91% after 28 days. Due to partial adsorption of the test item to the sludge, the DOC measurements cannot be used to conclude with certainty on the degradability of the substance Based on CO2 evolution data (mineralization data), Sulfosuccinic (isoFA C10)diE, Na showed significant mineralization during the incubation period since average 58% of the test material was mineralized within 28 days. Sulfosuccinic (isoFA C10)diE, Na can thus be regarded at least as inherently, primarily biodegradable (OECD guideline for testing of chemical, Annex 1 Section 3, Part 1, April 2005).

Results with reference substance:

DOC (elimination):
Biodegradation of the reference substance reached 97 % within 14 days of incubation. Hence the test is rendered valid.

Mineralization (CO2 production):
The procedure control (one replicate) for the mineralizsation part of the test (CO2 evolution measurements) did not yield reasonable results after days 7, most likely due to system leakage. Until then 51% of the reference substance had been mineralized. The S-shape of the duplicated mineralization curves however indicate that the test item systems were operated without leakage and thus the mineralization results are considered valid.

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The results show evidence for inherent biodegradablability. At test initiation, 55% of the substance disappeared from the DOC fraction after addition of the substance to the inoculum, most likely due to binding to the sludge. Thereafter, the test system equilibrated during the subsequent 11 days. From then onwards, S-shape elimination (i.e. biodegradation) was found, i.e. 75% were eliminated between day 11 and day 28, i.e. within 17 days of incubation. Total elimination during 28 days was average 91%. The results of CO2 evolution show that a significant part of the elimination was due to mineralization of the test substance since 58% CO2 were produced by the end of the test.

Executive summary:

The study was performed under GLP and according to appropriate methods to address the endpoint of inherent biodegradation. The biodegradability of Sulfosuccinic (isoFA C10)diE, Na (test item) exposed to microorganisms derived from non-adapted, activated sludge of a municipal sewage treatment plant „Weidhölzli“ in Zürich was investigated for 28 days in the dark under aerobic static exposure conditions and a temperature of 22 ± 1 °C using the Manometric Respirometry Test OECD 301F. For that purpose, 113 mg/L test substance (47,5 mg/L TOC) were incubated in an inocculum, which had been prepared with standard OECD medium and 200 mg/L dry mass of non-adapted, washed sludge. For that purpose, 2.2 liter closed glass bottle containing 2 L of test solution were used. They were aerated with CO2-free air and fitted to gas-absorption bottles containing 125 ml of 0.13 M KOH during the test. Three test groups were set-up : (1) Test Group with test substance concentration 113 mg/L (47.5 mg/L TOC, in triplicate), (2) Blank control (in triplicate) and (3) Procedural control diethylene glycol 110 mg/L (50 mg/L TOC, in triplicate). During the test DOC (substance elimination) was measured prior to and after sludge addition, 3 hours thereafter and after 1, 4, 7, 11, 14, 18, 21, 25, 27 and 28 days. In addition, CO2 (mineralization) was measured after incubation initiation (Day 0), and on days 7, 14, 21 and 28. DOC and IC (CO2) were measured by appropriate analytical equipment (TOC analyzer). The procedural control, diethylene glycol, showed 97% elimination based on DOC removal on day 14, thus confirming suitability of inoculum under test conditions (trigger for validity: 70%). Based on CO2 production investigation of the procedural control was erroneous from day 14 onwards (most likely due to system leakage between reactor and trapping system). However mineralization data are considered valid since the procedural control had already produced 51% CO2 by day 7, since mineralization curves showed continuous biodegradation (S shaped curves) and since elimination data show that the test system is suitable for this test. Hence these mineralization results are valid. Measurements prior to and after additional of the sludge show at the initiation, that average 55% of the test item was bound to sludge. During the first 11 days, the test system equilibrated and elimination showed a typical S-shape curve thereafter: Based on DOC measurements, Sulfosuccinic (isoFA C10)diE, Na was eliminated by average 91% at end of the incubation period. Due to partial adsorption of the test item to the sludge, the DOC measurements can however not be used to conclude with certainty on the biodegradability of the substance. Based on CO2 evolution data (mineralization data) however, Sulfosuccinic (isoFA C10)diE, Na showed significant mineralization during the incubation period since average 58% of the test material was mineralized within 28 days. Sulfosuccinic (isoFA C10)diE, Na can thus be regarded at least as inherently biodegradable (OECD guideline for testing of chemical, Annex 1 Section 3, Part 1, April 2005). The significant mineralization of 58% after 28 days of Sulfosuccinic (isoFA C10)diE, Na (based on CO2 evolution and calculated as % ThCO2) confirms that a significant part of the test item was ultimately biodegraded; the rest was either adsorbed onto the sludge, or incorporated in the bacterial biomass.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Conduct of Study: April 2013 (Test initiation April 25 2013)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

GLP compliance:

yes

Remarks:

The study was conducted under ISO/EC 17025. This certification standard is accepted according to 648/2004/EEC to assess the biodegradation of surfactants

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Non-adapted activated sludge from the aeration tank of the STP Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant. 30 mg/l dry matter in the final mixture.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: Aerobic conditions. The sludge was not fed. Environmental conditions during storage not reported. Ambient laboratory conditions assumed.
- Storage length: The activated sludge was used after sampling from the treatment plant without adaptation. However, the sludge was pre-conditioned for 3 days to reduce the amount of CO2 produced by the blank controls.
- Preparation of inoculum for exposure: The activated sludge was washed twice: One time with tap water and one time with mineral medium. After centrifugation, the sludge was suspended in test medium, at a tenfold concentration of the final concentration to be achieved for the test.
- Pretreatment: No data.
- Concentration of sludge: 30 mg dry matter/L.
- Initial cell/biomass concentration: Not provided.
- Water filtered: No data are provided in the report.
- Type and size of filter used, if any: No data.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

other: TOC

Initial conc.:

44.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD 301 medium. Aerobic mineral salts medium acc. to OECD 301 prepared with ultrapure water (conductivity: <1.5 μS/cm; DOC: <0.5 mg/l).
- Test temperature: 22 +/- 2 °C.
- pH: At test initiation, pH was adjusted to pH 7.4 (± 0.1) with NaOH or HCl, where necessary. At the end of exposure, the pH values of both inoculum blanks and the procedure control were 7.2, respectively. The pH values of the test units and the toxicity control were 7.3 and 7.4, respectively.
- Aeration of dilution water: Yes. The test vessels were stirred thoroughly and aerated with synthetic CO2-free air for a test period of 28 days. The air leaving the individual vessels was passed through gas-absorption bottles filled with KOH.
- Suspended solids concentration: 30 mg/L dry mass per testing unit.
- Continuous darkness: yes. Temperature-controlled dark room.

TEST SYSTEM
- Culturing apparatus: 2500 ml closed glass bottle containing a total volume of test solution of 2000 ml; aerated with CO2-free air and fitted to gas-absorption bottles containing 125 ml of 0.13 M KOH.
- Number of culture flasks/concentration: Eleven.
Test material (44.9 mg/L, 20 mg/L TOC, 3 replicates).
Blanc control (3 replicates).
Reference substance ( 20 mg/L TOC, sodium benzoate, one replicates).
Abiotic control (0.2 mM HgCl2, one replicate).
- Method used to create aerobic conditions: Stirring.
- Measuring equipment: TOC analyser.
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics if used: CO2 as produced by the inocculum was absorbed by 125 mL of 0.13 M KOH, which had been exposed in gas-washing bottles connected serially to the test vessels.

SAMPLING
- Sampling frequency: Days 0, 1, 5, 7, 11, 13, 19, 21, 26 and 28 (CO2 production, TOC measurements).
- Sampling method: Aliquot samples were removed from duplicate gas washing bottles to measure TOC.
- Sampling frequency: Day 28 (DOC measurement).
- Sampling method: One sample was sacrificed after acidification to measure DOC (ultimate biodegradation) on day 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (three replicates).
- Abiotic sterile control: Yes (sterilized with 0.2 mM HgCl2, one replicate).
- Procedure control: 29 mg/L TOC, sodium benzoate.
- Toxicity control: Yes (20 mg/L TOC test substance and 20 mg/L TOC sodium benzoate, one replicates).

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 (CO2 evolution)

Value:

45

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

86

Sampling time:

28 d

Details on results:

The substance was mineralized within 28 days by average 45,2%, mean of 47,9% and 42,5%. Trigger for ready biodegradation is 60% (according to 648/2004/EEC). Based on DOC measurement on day 28, 86% of the test substance has been eliminated. The substance was not toxic to the activated sludge, since 56,2% of the reference substance was degraded within 13 days in the presence of the test substance (trigger is 25%). The abiotic sterile control produced maximum 1.1 mg IC/L. Hence the test material was abiotically not degraded.

Results with reference substance:

Biodegradation of the reference substance reached 88.6% after 13 days of exposure to the sludge.

Validity criteria fulfilled:

yes

Remarks:

However deviation between test item replicates was 21% (validity trigger: 20%)

Interpretation of results:

other: not readily biodegradable

Conclusions:

In conclusion, the substance is not readily biodegradable according to the criteria of OECD 301 TGD: Average biodegradation (45,2%, mean of 47,9% and 42,5% based on CO2 evolution is below the trigger value of 60% (10 day window not applicable). Though 86% had been eliminated by day 28, ultimate biodegradation is partial, but significant. A plateau had not been reached on day 28. The 10% generic trigger value for biodegradation was reached within 11 days after test initiation.

Executive summary:

In a valid GLP study according to OECD 301B the test item was aerobically exposed at a concentration of 44,9 mg/L test substance (20 mg/L TOC) to standard OECD 301 mineral medium and 30 mg dry mass/L activated, non-adapted sludge. The inocculum was obtained from a domestic STP. Exposure was for 28 days in the dark at pH 7.2 to pH 7.4, under stirring conditions and between 20 and 24°C. Eleven closed bottle glass vessels with a total volume of 2.5 L each were used and constantly aerated with CO2 free air. Each test vessel had received a total volume of 2 L inocculum and mineral medium. Five test groups were incubated: Test item (20 mg/L TOC, three replicates), blank control (three replicates), reference substance sodium benzoate (20 mg /L TOC, one replicate), a toxicity control (20 mg/L TOC test substance plus 20 mg/L TOC reference substance, one replicate) and an abiotic control. CO2 as produced by the inocculum was absorbed by 125 mL of 0.13 M KOH, which had been exposed in gas-washing bottles connected serially to the test vessels. Trapped carbon dioxide was determined as inorganic C by means of an appropriate C analyzer. For that purpose, duplicate samples were measured. Biodegradation was determined on days 0, 1, 5, 7, 11, 13, 19, 21, 26 and 28. On day 28, one replicate was used to measure DOC, in order to determine elimination of the substance. The results show that all validity criteria of the test guideline were met. Hence the results obtained for the test item are valid. The substance was mineralized within 28 days by average 45,2%, mean of 47,9% and 42,5%. Trigger for ready biodegradation is 60% (according to 648/2004/EEC). Based on DOC measurement on day 28, 86% of the test substance has been eliminated. This indicates that ultimate biodegradation was only partial. However, a CO2 evolution plateau had not been reached on day 28. In conclusion, the substance is not readily biodegradable according to the criteria OECD 301 guideline. The 10% generic trigger value for biodegradation was reached within 8 days after test initiation. The substance was not toxic to the activated sludge, since 56,2% of the reference substance was degraded within 13 days in the presence of the test substance (trigger is 25%). The abiotic sterile control produced maximum 1.1 mg IC/L. Hence the test material was abiotically not degraded.

Description of key 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, 45% (43-48%) of the test material was ultimately degraded.

Conclusion: The substance is not readily biodegradable.

 

A further study for ready biodegradability is available. The test was performed according to OECD 301 F and is valid according to the guideline criteria. After incubation for 28 days, 26% (23-29%) of the test material was ultimately degraded.

Conclusion: The substance is not readily biodegradable.

 

A further 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, 24% of the test material was ultimately degraded. Conclusion: The substance is not inherently biodegradable.

 

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, 14 ± 6% (7-14-22%) of the test material was ultimately degraded. After incubation for 60 days, 64 ± 11% (51-62-79%) of the test material was ultimately degraded. Set-up 2: After incubation for 28 days, 26 ± % (21-24-28-31%) of the test material was ultimately degraded. After incubation for 60 days, 78 ± 14% (60-70-87-96%) of the test material was ultimately degraded.

 

A study supporting the key study for ready and enhanced biodegradability is available. The test was performed according to OECD 301 F in triplicate and is valid according to the guideline criteria (difference between replicates at the end of the 10-day window < 20%). However, results obtained on day 28 are not reliable, since the difference between extremes was > 20%.  After incubation for 60 days, 78 ± 24% (56-74-104%) of the test material was ultimately biodegraded.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria