Registration Dossier

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11.06.2012 - 11.07.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
yes
Remarks:
No test item specific analysis performed
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water (see Section 3.4.1) to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of two days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 100 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
73 other: mg O2/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The following modifications versus OECD 302C were made: activated sludge from one source was used, the activated sludge was not fed during the holding period of two days, the test was run at 22 °C, the test water composition was slightly changed, and only the biological oxygen demand (BOD) was monitored, no test item specific analysis was performed.
Reference substance:
benzoic acid, sodium salt
Test performance:
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg (ThOD).
In the procedure controls, the reference item (sodium benzoate) was degraded by an average of 76.6% and 87.7% by Exposure Days 7 and 14, respectively, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 90.1%.
Parameter:
% degradation (O2 consumption)
Value:
70.2
Sampling time:
28 d
Remarks on result:
other: as mean of duplicates
Results with reference substance:
The results are considered valid since the following criterion is met:
The percentage biodegradation of the reference item sodium benzoate calculated from the oxygen consumption were higher than 40% of the ThOD after 7 days and higher than 65% of the ThOD after 14 days of incubation.

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.44 mg O2/mg test item. The biochemical oxygen demand (BOD) of Neo Heliopan HMS in the test media significantly increased from test start until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of Neo Heliopan HMS amounted to 70.2% of ThOD. The test was terminated before the biodegradation of the test item reached a plateau.

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
In an OECD302C guideline test the substance reached 70.2% biodegradation and thus is considered inherently, ultimately biodegradable.
Executive summary:

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.44 mg O2/mg test item. The biochemical oxygen demand (BOD) of Neo Heliopan HMS in the test media significantly increased from test start until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of Neo Heliopan HMS amounted to 70.2% of ThOD. The test was terminated before the biodegradation of the test item reached a plateau. Consequently, according to the OECD Guideline for Testing of Chemicals, Neo Heliopan HMS can be regarded as inherently, ultimately biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2017 to 23 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992, enhanced to 60 days with monitoring of metabolites
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: 50100700
- Purity: 99.8%
- Appearance: Colorless to light yellow, clear liquid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source: Municipal sewage treatment plant, 31137 Hildesheim, Germany
Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

- Pre-treatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2.5 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with C02-free air for 5 days. 10 mL/L were used to initiate inoculation.

- Colony Forming Units of the Inoculum: Colony forming units (CFU) o f the inoculum were determined prior to test start by standard dilution plate count: 1.31 x 109CFU/L. The CFU concentration of the inoculum corresponds to approx. 1.31 x 107 CFU/L in the final test solution.
Duration of test (contact time):
60 d
Initial conc.:
25 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The inoculated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and inoculum, was prepared in a measuring flask.

- Test temperature: 20°C
- Light regime: Darkness
- pH: 7.63 - 7.73 (with test item)
- pH adjusted: no

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (500 mL)
- Number of culturing flasks per concentration: 12 for test item
- Method used to create aerobic conditions: Continuous stirring
- Measuring equipment: Oxitop measuring heads

SAMPLING
- Sampling frequency: 360 measuring points during 60d period, primary degradation was determined at day 0, 14, 28 and 60 via analytical monitoring
- Sample storage before analysis: Extracted samples and the extracted test medium were stored at 6 ± 2°C. Prepared samples were stored at ambient temperature in an autosampler rack prior to analysis. The stability of the fortified samples was verified after extraction for 36 days.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 inoculum blanks
- Abiotic sterile control: no
- Toxicity control: 2 toxicity controls
- Functional control: 1 functional control with reference item sodium benzoate
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
60 d
Remarks on result:
other: mean value
Parameter:
% degradation (O2 consumption)
Value:
34
Sampling time:
28 d
Remarks on result:
other: mean value
Details on results:
The mean oxygen depletion in the inoculum control was 4.5 mg oxygen/L on day 28 and 10.7 mg
O2/L on day 60.
In order to check the activity of the test system sodium benzoate was used as functional control.
The pass level > 60% was reached within 4 days. The biodegradation reached a maximum of 100% degradation on day 17.
In the toxicity control containing both test and reference item 60% degradation occurred within 15
days. After 28 days the biodegradation was 65% and after 60 days it came to 84%. The degradation of the reference item was not inhibited by the test item.
The mean of replicates of the test item reached the 10% level (beginning of biodegradation) on day 13 and the 60% pass level on day 56. The mean biodegradation on day 60 was 62%.
In general, the biodegradation curves show a typical biphasic nature which might indicate the subsequent degradation of the two moieties of the test item. While the lag phase for the first step was quite homogenous and approx, between day 8 and 15, the lag phase for the second step was quite different between the replicates. While some replicates continued with the biodegradation process after a few days, other replicates remained on the level of the first step until test end. After 60 days, the majority (n=5) of the remaining 8 replicates reached the 60% threshold and one replicate was slightly below the pass level criterion (56%), but still increasing. This indicates, that under ideal conditions (e.g. bioavailability, competent degraders in the inoculum) the test item is inherently biodegradable.

Primary degradation:
The results of this enhanced biodegradability test according to OECD 301F showed a clear bi-
phasic biodegradation pathway of Neo Heliopan® HMS. Specific analysis of the parent compound and metabolites proved a very fast primary degradation after a short lag phase. Already after 14 days, the parent compound was no longer found, or found only in traces.
The firs t step in the degradation pathway is the cleavage of the ester bond resulting in Salicylic
acid and 3,3,5-Trimethylcyclohexanol. The biodegradation of the Salicylic acid (not considered
during analysis) is supposed to form the first mineralization step of the degradation pathway of the test item. This is supported by the finding that a derivative of 3,3,5-Trimethylcyclohexanol,
3,3,5-Trimethylcyclohexanone, can be found during samplings after 14, 28 and 60 days.
After 28 and 60 days, the respective replicates were showing considerable amounts of 3,3,5-
Trimethylcyclohexanone. 3,3,5-Trimethylcyclohexanol was not detected during the course of the
study and might be considered to be rather short-lived.
Comparing the samples, the first mineralization step was followed by a further lag phase, whose length varied between the samples, before the second mineralization step was likely to commence.
Replicates that had already passed the second step in mineralization showed degradation levels above the pass level criterion of 60% or nearly 60% (only in one replicate) at the test end. The complete mineralization of the test item after the second mineralization step was confirmed by analytical monitoring of 3,3,5-Trimethylcyclohexanone. No residual amounts of 3,3,5-Trimethylcyclohexanone have been detected in these samples. Summarizing all these findings leads to the conclusion that the test item is not persistent and will be degraded to metabolites
which will be completely mineralized by microorganisms. This assumption can be supported
by literature data for the two most important metabolites salicylic acid and 3,3,5-Trimethylcyclohexanone, which showed that both substances are readily biodegradable [ECHA database].

The toxicity control showed that the test substance is not inhibitory at a concentration of 25 mg/L.
Results with reference substance:
The pass level for ready biodegradation (> 60% degradation) was reached within 4 days. The validity criterion that the degradation should be a 60% after 14 d was fulfilled. Stages of biodegradation:
The adaptation phase changed to degradation phase within 2 days (degradation > 10%) and the biodegradation came to a maximum of 100% on day 17.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The prolonged ready biodegradability test (OECD 301F) proved that the test substance is inherently biodegradable.
Furthermore, based on the proposed degradation pathway and the measured metabolites, it could be shown that the formed metabolites are not persistent.
Executive summary:

The prolonged ready biodegradability test according to OECD 301F over 60 days, proved that the test item is not readily (34% after 28 days), but inherently biodegradable (62% after 60 days).
Specific analysis of the parent compound and metabolites proved a very fast primary degradation (cleavage of the ester bond resulting in salicylic acid and 3,3,5-Trimethylcyclohexanol) after a short lag phase (only traces of test item after 14 days). The first mineralisation step was followed by a further lag phase before the second mineralisation step was likely to commence. The complete mineralization of the test substance after the second mineralization step was confirmed by analytical monitoring of 3,3,5-Trimethylcyclohexanone. No residual amounts of 3,3,5-Trimethylcyclohexanone have been detected in these samples. Summarizing all these findings leads to the conclusion that the test substance is not persistent and will be degraded to metabolites which will be completely mineralized by microorganisms. 


 

Description of key information

The test substance is inherently biodegradable and not persistent. It will be degraded to metabolites which are not persistent and will be completely mineralized by microorganisms. 

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Three reliable biodegradation tests are available: Two ready biodegradability test according to OECD 301 F and an inherent biodegradability test based on OECD 302C.


In the key study on ready biodegradability with non-adapted activated sludge according to OECD 301F the test duration was prolonged to 60 days. The degradation was followed by measuring the oxygen consumption and additionally metabolites were analysed at day 14, 28 and 60 of the study. The mean degradation after 60 days was 62% and thus, the test substance can be considered as inherently biodegradable. Specific analysis of the parent compound and metabolites proved a very fast primary degradation (cleavage of the ester bond resulting in salicylic acid and 3,3,5-Trimethylcyclohexanol) after a short lag phase (only traces of test item after 14 days). The first mineralisation step was followed by a further lag phase before the second mineralisation step was likely to commence. The complete mineralization of the test substance after the second mineralization step was confirmed by analytical monitoring of 3,3,5-Trimethylcyclohexanone. No residual amounts of 3,3,5-Trimethylcyclohexanone have been detected in the samples at the end of the study (60 days) when the second step in mineralisation occured. This shows that the test substance and its metabolites are not persistent. The degradation of the reference item was not inhibited by the test item.


In a second experiment the inherent biodegradability was investigated in a test based on the OECD 302C method. No test item specific analysis was performed. The test was terminated after 28 days, before the biodegradation of the test item reached a plateau. The substance exhibited a biodegradation of 70.2% within 28 days in this test. Therefore the substance can be regarded as inherently and ultimately biodegradable.