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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
29 d
Remarks on result:
not determinable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-30 to 2014-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
version adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge, micro organisms from a domestic waste water treatment plant
- Origin: sewage plant Neustadt, Germany
- Method of cultivation: The sludge was filtrated, washed twice with tap water, then washed with and re-suspended in test medium. It was aerated until use.
Duration of test (contact time):
28 d
Initial conc.:
51.7 mg/L
Based on:
test mat.
Remarks:
corresponds to 20.1 mg organic C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: According to the OECD TG 301D (In solution b) CaCl2*2H2O was used and in solution d) 0.4g Na2EDTA were added).
- Test temperature: 21.4 – 24.0 °C
- pH: 6.9 – 7.5 (on day 28)
- pH adjusted: none
- Aeration of dilution water: for 72 h
- Suspended solids concentration: 24.9 mg/L

TEST SYSTEM
- Culturing apparatus: glass flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with purified, CO2-scrubbed, moistened air
- Measuring equipment: CO2 was measured by IC using a carbon analyser
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: scrubber flasks

SAMPLING
From each front scrubber flask, ten samples were taken in order to determine the emitted CO2 (on days 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Test flasks: 2
- Abiotic control: 1
- Toxicity control: 1
Reference substance:
aniline
Test performance:
Sample Preparation: The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its TOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined. The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2. The test was performed with a nominal start concentration of 20 mg organic carbon/L.

CO2 Determination: Analyses of the emitted CO2 were made by IC measurement using the carbon analyser. Each sample was measured at least in duplicate.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
29 d
Remarks on result:
not determinable
Details on results:
Ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test item cannot be considered as “readily biodegradable”. Degradation missed 60% within 28 days, too. Therefore, the test item is not considered as “biodegradable within 28 days”, either.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.
Results with reference substance:
The reference item aniline was sufficiently degraded to a mean of 68.1 % after 14 days, and 76.9 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item, a biodegradation of 35% was observed after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory on the activated sludge microorganisms.

Validity


All validity parameters and values are presented in the following table:










































Parameter



Criterion



Found



Assessment



IC content of test item solution in medium



< 5% of TC



< 1 %



valid



CO2 emitted by the controls



< 70 mg/L



13.2 mg/L



valid



Difference within replicates



< 20%



< 1 %



valid



Degradation of positive control > 60%



< 14 days



9 days



valid



Degradation in the toxicity flask on day 14



> 25%



35.0 %



valid



Degradation Values


In the following table, the percentage biodegradation is presented:


Table 1: Degradation values in %




















































































































Day



Positive


Control


1



Positive


Control


2



Positive


Control


Mean



Test 1



Test 2



Test


Mean



Abiotic


Control



Toxicity


Control



2



-0.8



-0.2



-0.5



0.1



1.3



0.7



0.0



0.1



4



13.2



23.2



18.2



2.0



1.3



1.6



0.1



8.6



7



42.1



48.2



45.1



3.4



1.8



2.6



0.5



22.2



9



65.9



64.0



65.0



3.2



1.2



2.2



0.3



29.3



11



68.1



67.6



67.9



3.6



1.7



2.7



1.0



30.6



14



67.9



68.4



68.1



2.1



2.7



2.4



0.7



35.0



18



77.1



78.5



77.8



3.3



2.9



3.1



1.0



39.8



23



74.8



71.0



72.9



2.1



2.3



2.2



0.9



38.5



29



79.2



74.6



76.9



1.3



0.5



0.9



0.6



39.8



 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In a study according to OECD TG 301 B under GLP, the substance was found to be not readily biodegradable.
Executive summary:

The ready biodegradability of the test item was investigated in a study according to OECD TG 301 B and under GLP using a test item concentration of nominal 20 mg organic carbon/L (corresponding to 51.5 mg test item/L). The biodegradation was followed by the CO2-evolution. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Activated sludge was used as inoculum (concentration in the test 24.9 mg dry matter/L). The test item in a mineral medium was inoculated and incubated under aerobic conditions in the dark and the test was left running for 28 days. The amount of DOC in the test solution due to the inoculum was kept as low as possible compared with the amount of organic carbon due to the test item. Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without test item. Measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. All validity criteria were met. The reference item aniline was sufficiently degraded to a mean of 68.1 % after 14 days, and 76.9 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a biodegradation of 35% was observed after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory on the activated sludge microorganisms. No degradation was detected within the 10-day window. The test item emitted almost no CO2, so that the biodegradation after 29 days was determined as 1%. Ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test item cannot be considered as “readily biodegradable”. Degradation of the test item missed 60% within 28 days, too. Therefore, the test item is not considered as “biodegradable within 28 days”, either.

Description of key information

This endpoint is covered by an analogue approach.


In a study according to OECD TG 301 B under GLP, the source substance was found to be not readily biodegradable (reference 5.2.1-1). Thus, reading this result across, also the target substance is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

No biodegradability study with the test item was available, thus, a read across to a structural analogous material was done instead. Please refer to section 13 for justification of the read-across.


The ready biodegradability of the test item was investigated in a study according to OECD TG 301 B and under GLP using a test item concentration of nominal 20 mg organic carbon/L (corresponding to 51.5 mg test item/L). The biodegradation was followed by the CO2-evolution. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Activated sludge was used as inoculum (concentration in the test 24.9 mg dry matter/L). The test item in a mineral medium was inoculated and incubated under aerobic conditions in the dark and the test was left running for 28 days. The amount of DOC in the test solution due to the inoculum was kept as low as possible compared with the amount of organic carbon due to the test item. Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without test item. Measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. All validity criteria were met. The reference item aniline was sufficiently degraded to a mean of 68.1 % after 14 days, and 76.9 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a biodegradation of 35% was observed after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory on the activated sludge microorganisms. No degradation was detected within the 10-day window. The test item emitted almost no CO2, so that the biodegradation after 29 days was determined as 1%. Ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test item cannot be considered as “readily biodegradable”. Degradation of the test item missed 60% within 28 days, too. Therefore, the test item is not considered as “biodegradable within 28 days”, either.