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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
60 days biological part of the study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study describes the qualitative and quantitative analysis of the biodegradation study 2014a. The analytical results were not performed under GLP:
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The biodegradation study was performed under OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test). For the analyitical investigationsinternal laboratory protocols were used.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
60 d
Initial conc.:
500 µg/L
Details on results:
Samples of day 0, 35 and 42 were directly deep-frozen and stored until analysis. Just prior to analyis, samples were de-frozen and directly injected into a LC/MS/MS System using a standard gradient water-HCOOC + Acetonitrile:
HPLC: HP1290 (Agilent)
MS/MS: API 5500 (Applied Biosystem)

As the concentration in the test solution were rather low, possible structures from the Minnesota Prediction Program and from a publication were taken and the tentative masses (M+H) measure by selected ion monitoring.

Two metabolites could be identified and possible structures were defined: N-(2((2-aminophenyl)disulfanyl)phenyl)benzamide and N-(1E)-4-oxo-2-sulfanylcyclohexa-2,5-dien-1-yoliden)benzanmide.

Remaining parent substance was analysed and quantified:
day 0 21 µg/L
day 35 0.8 µg/L
day 42: 3.4 µg/L
These results are probably underestimated as the start concentration on day 0 was 500 µg/L. It is suspected that considerable parts of the substance were adsorbed to biomass or to the glass walls.

Two metabolites could be identified and possible structures were defined: N-{2-[(2-aminophenyl)disulfanyl]phenyl}benzamide and N-[(1E)-4-oxo-2-sulfanylcyclohexa-2,5-dien-1-ylidene]benzamide. The structural formula are attached to the IUCLID dataset.

Remaining parent substance was analysed and quantified:

day 0 21 µg/L

day 35 0.8 µg/L

day 42: 3.4 µg/L

These results are probably underestimated as the start concentration on day 0 was 500 µg/L. It is suspected that considerable parts of the substance were adsorbed to biomass or to the glass walls.

Interpretation of results:
not readily biodegradable
Conclusions:
The biological part of the work showed some biodegradation and analysis work has been performed to evaluate the potential metabolites.
The work was ultimately inconclusive in terms of quantification, but does lead to the identification of two metabolites.
At the time of the initial testing, the low recovery of the parent substance at the start of the study (day 0 analysis) was considered to be due to poor solubility and potential adsorption to surfaces, but as a result of later work (See 5.2.2), hydrolysis is considered to lead to rapid cleavage of the sulphur bridge.
It is very likely that a combination of biotic, abiotic and adsorption/desorption processes are taking place, but it can be concluded that the parent substance will be transformed to smaller fragments that may themselves be more likely to degrade.
Executive summary:

Report conclusions:

A biodegradation rate of 27% at day 42 can be explained by partial degradation of the substance yielding metabolites 1 and 2.

Metabolite 1 is formed by oxygen consumption (hydroxylation and oxidation).

Metabolite 2 is formed by cleavage of the parent substance yielding benzoic acid which is known to be readily biodegradable. Thus benzoic acid is also responsible for oxygen consumption during the test. However, no information is available about the percentage of metabolism as no quantification of the metabolites was possible.

The parent substance itself could be found in all samples which were investigated.

The starting concentration was 500 µg/l which is more than 10 fold above the water solubility. The low water solubility together with a high adsorption potential (e.g. to biomass or glass walls) may explain why the biodegradation does not continue for completion and stops in the range of 20 to 30 %.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
60 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guidline study etended to 60 days performed on test material.
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
(2008)
Deviations:
yes
Remarks:
on sponsor’s request the required quantity of the test item was dropped down to 0.5 mg/L and the duration of the test was prolonged up to 60 days
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
(1992)
Deviations:
yes
Remarks:
on sponsor’s request the required quantity of the test item was dropped down to 0.5 mg/L and the duration of the test was prolonged up to 60 days
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Test organisms
- Type: mixed population of aquatic microorganisms
- Origin: secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2014-06-25
- Pre-treatment: separation of coarse particles by filtration, aeration of the resulting inoculum for 1 day
- Effluent concentration of reaction mixture: 2.6 mL/L (3 mL/1.16 L)
Duration of test (contact time):
60 d
Initial conc.:
0.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The method assessed the ready biodegradability of a test item under aerobic conditions at 22 +/- 2 °C in the dark. On sponsor’s request the test lasted for 60 days. During this period, the biodegradation was determined based on the reduction of dissolved oxygen (BOD). A measured volume of inoculated mineral medium, containing a known concentration of 0.5 mg test item/L (usually 2-10 mg/Litre), to give usually 5-10 mg ThOD/Litre as the nominal sole source of organic carbon in a closed flask was applied. The degree of biodegradation was calculated by the amount of oxygen taken up by the test item (corrected for that in the blank inoculum control) as a percentage of ThOD (theoretical oxygen demand).

The following flasks were used:
- Test suspension (30 flasks)
A measured volume of mineral medium containing a known concentration of the test item (as the nominal sole source of organic carbon) and inoculum.
- Reference control (20 flasks)
In order to check the procedure, the reference compound sodium benzoate was tested in parallel to the normal test runs.
- Inoculum blank (20 flasks)
A measured volume of mineral medium containing only inoculum.

A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms (according to data of the sponsor).

Degradation was followed by analysis of dissolved oxygen over a 60-day period. The amount of oxygen taken up by the microbial population during biodegradation of the test item, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of ThOD.

On sponsor’s request he test lasted for 60 days.
Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, all determinations were carried out in duplicate.

EXPOSURE CONDITION:
- Test volume: 1.16 L
- Test apparatus: OxiTop System (WTW)
- Mixing: 1 magnetic stirrer per test vessel
- 60 days incubation at 22 +/- 1 °C
Reference substance:
benzoic acid, sodium salt
Remarks:
(purity: 99.7 %, batch no.: 1438955, 2.9 mg/L)
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
6
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
27
Sampling time:
42 d
Parameter:
% degradation (O2 consumption)
Value:
17
Sampling time:
60 d
Details on results:
More results of the weekly measurement are found in "any other information on results".
Parameter:
COD
Value:
2.208 mg O2/g test mat.
Results with reference substance:
Kinetic of reference substance (degradation):
75 % after 7 days
69 % after 14 days
81 % after 21 days
81 % after 28 days
82 % after 60 days

Results of degradation of the test item

 Test item  7 d  14 d  21 d  28 d  35 d  42 d  49 d  56 d  60 d
 % degradation  14  6  5  17  98*  27  20  22  17

* results could not be validated by instrumental analysis.

After 60 days the pH values of different flaks are in the range of 6.8 - 7.2.

Validity criteria fulfilled:
yes
Remarks:
(- Ready biodegradability of reference compound ≥ 60 percent whitin 14 days. - At the end of the test, at the plateau, or the end of the 10-d window, biodegradation in parallels with test item did not differ by more than 20 percentage points.)
Interpretation of results:
other: not readily biodegradable
Conclusions:
Within 60 days, a degradation rate of 17 % was determined. The test item is considered to be "Not Readily Biodegradabable". The reference compound sodium benzoate showed 69 % after 14 days.
Executive summary:

To test for its biodegradability potential, the substance was incubated for 60 days in continuously stirred 1.16 L closed flask (in duplicate) in the dark with an inoculum originally collected from a local, predominantly municipal wastewater treatment plant. In this assay, biodegradation was estimated by biological oxygen demand (BOD) over time. BOD was measured daily by a manometer. The incubation temperature was 22 ± 2 °C, pH was 6.8 - 7.2. The concentration of innoculum was 30 mg /L and the one of the substance was 0.5 mg/L. Degradation was calculated by substracting the amount BOD in the negative (innoculum only) control from that in the substance or positive control at any given time point and divided by the chemical oxygen demand (COD) or thereotical oxygen demand (ThOD). The 60-day degradation was 17% and for the positive control (with reference substance) the biodegrdation was 69 %. In conclusion the substance is not readily biodegradable.

Description of key information

The biological part of the key 60 day study showed some biodegradation of up to 27% and analysis work has been performed to evaluate the potential metabolites.

The work was ultimately inconclusive in terms of quantification, but does lead to the identification of two metabolites. 

At the time of the initial testing, the low recovery of the parent substance at the start of the study (day 0 analysis) was considered to be due to poor solubility and potential adsorption to surfaces, but as a result of later work (See 5.2.2), hydrolysis is considered to lead to rapid cleavage of the sulphur bridge.

It is very likely that a combination of biotic, abiotic and adsorption/desorption processes are taking place, but it can be concluded that the parent substance will be transformed to smaller fragments that may themselves be more likely to degrade.

It is concluded that the parent substance is not persistent, but may lead to the formation of lower molecular weight transformation products that are themselves also poorly biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

Other tests show 24% degradation after 28 days, considered to be limited by poor solublity in the key study.

An earlier supporting study resulted in 0% biodegradation over 28 days when performed at 100 mg/l. There appeared to be no toxicity, but as the positive control group showed very slow degradation, it may be that the innoculum was not very active. Again, solublity may have limited the ability to biodegrade.