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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-10-18 to 2015-02-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This substance is a natural extract. Natural extract can be gum, resinoid, concrete or absolute. This natural extract belongs to the group of Natural Complex Substances (NCS): UVCB sub-type 3, where the source is biological, and the process is refinement (ECHA Guidance on Identification and naming of substances under REACH, version 2.1 – May 2017, Section 4.3.1 and EFEO/IFRA Guidelines on substance identification and sameness of natural complex substances (NCS) under REACH and CLP, version of August 5, 2015).
The source substance (Cistus concrete) and the registered substance "Absolute of Cistus ladaniferus (Cistaceae) obtained from cistus concrete by heat
treatment and ethanol extraction" are obtained from the same botanical source: Cistus ladaniferus (Cistaceae). The registered substance is obtained from the source substance, Cistus concrete. Thus, Cistus concrete contains all the constituents available for the extraction of the registered substance. Therefore, the compositions, and then the expected behaviour are covered by Cistus concrete data. Therefore, we consider as reliable the read across from Cistus concrete dossier to this dossier.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
EEC Publication No. L 142/496, May 2008
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source of inoculum/activated sludgerom a domestic waste water treatment plant was supplied by the sewage plant Rossdorf, Germany.
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and centrifuged again. This procedure was done three times and aerated overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water. This suspension was used for the experiment.
Duration of test (contact time):
60 d
Initial conc.:
226.9 mg/L
Based on:
ThOD
Remarks:
corresponding to a loading rate of test material of 82.6 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
Pressure measurements
Details on study design:
TEST CONDITIONS
Reconstituted Test Water: Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with deionised water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with deionised water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with deionised water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with deionised water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) during storage, one drop of concentrated HCl per litre was added. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with deionised water.

Preparation of the Test Solutions:
Cistus Concrete
1) test item: 20.3 mg, Reference item: 0 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL
2) test item: 20 mg, Reference item: 0 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL
Inoculum Control
3) test item: 0 mg, Reference item: 0 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL
4) test item: 0 mg, Reference item: 0 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL
Procedure Control
5) test item: 0 mg, Reference item: 20.1 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL
Abiotic Control
6) test item: 20.2 mg, Reference item: 0 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 0 mL, Test water: 239.6 mL, Final volume = 244 mL
Toxicity Control
7) test item: 20.3 mg, Reference item: 19.9 mg, Silicon Oil AR20: 2.4 mL, Activated sludge: 2 mL, Test water: 239.6 mL, Final volume = 244 mL

- Solubilising agent: Silicon Oil AR20
- Test temperature: 22°C ± 1°C
- pH: 7.6 (measured at the start of the test) - 7.5 to 7.8 (measured at the end of the test)
- Continuous darkness: yes

TEST SYSTEM
The amounts of test item and reference item were directly weighed into the test flasks. For a better distribution of the test item also silicone oil AR20 was added at a concentration of about 1% in the test flasks.
The test item was weighed onto a cover glass and the cover glass was put into the test vessels and remained there for the whole incubation time.
- Replicates: Two replicates of test solution, two replicates of inoculum control, one replicate of procedure control, one replicate of abiotic control and one replicate of toxicity control were tested.

SAMPLING
The change of pressure in the test flasks was measured by means of a manometric method (BSB/BODSensor-System, Aqualytic Dortmund, Germany) each day.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No data.
Test performance:
Validity Criteria of the Study:
- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 30 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
- pH-Value: The pH-value of the test item flasks at the end of the test was 7.6 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
- Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation.
- Test Item: At the end of the experiment, the difference in replicate variation of the test item was 6%. The validity criterion was passed.
- Toxicity Control: In the toxicity control containing both, the test item and reference item, 39% biodegradation was noted within 14 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
Parameter:
% degradation (O2 consumption)
Value:
49
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
52
Sampling time:
60 d
Details on results:
The test item Cistus Concrete contains no nitrogen, therefore the evaluation of biodegradation has to be based ThODNH4.
The 10-day window started at day 3, when the mean biodegradation was 11%. At the end of the 10-day window at day 13, the mean biodegradation was 33%, after 28 days the mean biodegradation of Cistus Concrete was 49% (ThODNH4); the 10 day window criterion was not passed. The test was extended to 60 days, as no plateau was reached within 28 days. The biodegradation was 52% at the end of the test at day 60.
Therefore, Cistus Concrete is considered to be not readily biodegradable.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 78% after 14 days, to 80% after 28 days and to 78% after 60 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Biodegradation of the Toxicity Control:

In the toxicity control containing both, the test item and the reference item sodium benzoate, 39% biodegradation was noted within 14 days, 54% biodegradation after 28 days and 63% biodegradation after 60 days of incubation.

Thus, it can be assumed that the test item is not inhibitory to the aerobic activated sludge microorganisms.

Abiotic Control:

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use to correct the degradation of the test item and toxicity control.

pH-Values at the End of the Test:

Flask No. Treatment pH-value
1 Cistus Concrete 7.6
2 Cistus Concrete 7.6
3 Inoculum control 7.5
4 Inoculum control 7.5
5 Reference item (procedure control) 7.8
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Cistus Concrete is considered not readily biodegradable based on a mean degradation rate of 49% (THOD-NH4) at day 28. After prolongation of the test, the degradation rate reached 52% at day 60.
Executive summary:

Ready biodegradability of CISTUS CONCRETE was determined during this study. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 60 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The 10-day window started at day 3, when the mean biodegradation was 11%. At the end of the 10-day window at day 13, the mean biodegradation was 33%. After 28 days the mean biodegradation of Cistus Concrete was 49% (ThODNH4); the 10 day window criterion was not passed.

Therefore, Cistus Concrete is considered to be not readily biodegradable.

The test was extended to 60 days, as no plateau was reached within 28 days. The biodegradation was 52% at the end of the test at day 60.

The reference item sodium benzoate was sufficiently degraded to 78% after 14 days, to 80% after 28 days and to 78% after 60 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 39% biodegradation was noted within 14 days, 54% biodegradation after 28 days and 63% biodegradation after 60 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Description of key information

Based on a read-across from an experimental GLP study performed on the analogue substance Cistus concrete according to the OECD 301F guideline, the registered substance is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable study on the registered substance was not available. Therefore, in order to assess the ready biodegradability of the registered substance, the results from an experimental GLP study performed on the analogue substance Cistus concrete have been used.

In this study, the test item Cistus concrete was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days, according to the OECD 301F guideline, further extended to 60 days.

The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The 10-day window started at day 3, when the mean biodegradation was 11%. At the end of the 10-day window at day 13, the mean biodegradation was 33%. After 28 days the mean biodegradation of Cistus Concrete was 49% (ThODNH4); the 10 day window criterion was not passed.

Therefore, Cistus Concrete is considered to be not readily biodegradable.

The test was extended to 60 days, as no plateau was reached within 28 days. The biodegradation was 52% at the end of the test at day 60.

The reference item sodium benzoate was sufficiently degraded, thus confirming the suitability of the aerobic activated sludge inoculum used.

In addition, the test item was assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days in the toxicity control.

The validity criteria were successful and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.