Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2017 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 17. Jul. 1992
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
adopted 30. May 2008
Deviations:
yes
Remarks:
please refer to "Principles of the method if other than guideline"
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage.

Source: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) Date of collection: 12. Jan. 2018, batch no: 20180112.

Pre-Treatment: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 4900 mg of suspended solids/L.
Duration of test (contact time):
28 d
Initial conc.:
181.4 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its DOC was measured. The stock solution of the test item was prepared and its DOC 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.

Experimental Parameters
Flask volume 1500 mL
Apparatus blanks 2, containing mineral medium only
Blank Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing test item, mineral medium and inoculum
Abiotic control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration 25.0 mg/L
Temperature 19.5 – 21.2 °C
Duration 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L.

Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

Sampling
From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2 (see also chapter 8.3.1).
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

CO2 Determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month. After every start, quality control samples were measured.

Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
95
St. dev.:
3.3
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
97
St. dev.:
1.8
Sampling time:
28 d
Details on results:
Results for the Test Item
- The test item is considered as “readily biodegradable“.
- The degree of biodegradation reached 97 % after 28 days.
- The 10-day-window began on day 2, at its end, 95 % degradation were reached (graphic evaluation), surpassing the pass level of 60 % given in the OECD guideline.
- Because the test item is a mixture, the 10-day window has not to be taken into account. Therefore, regardless of the 10-day-window the test item is considered as “readily biodegradable within 28 days”.
- The abiotic degradation reached 5 %.

Degradation values in %

 Day Positive Control 1  Positive Control 2  Positive Control Mean  Test 1  Test 2  Test Mean  Abiotic Control  Toxicity Control 
 2  -0.4  0.0  -0.2  5.0  6.4  5.7  4.5  -0.2
 4  3.2  22.2  12.7  62.3  69.8  66.1  6.3  16.3
 7  57.1  57.7  57.4  75.1  76.1  75.6  5.1 61.8 
 9  80.5  80.1  80.3  89.2  98.9  94.1  5.3  77.9
 11  87.4  92.6  90.0  91.2  97.8  94.5  5.4  79.9
 14  95.2  96.3  95.8  92.3  98.6  95.5  5.2  84.3
 18  95.4  97.3  96.4  98.4  102.6  100.5  4.9  87.6
 23 102.6   101.0  101.8  96.7  100.6.  98.6  5.1  87.0
 29  100.8  101.3  101.1  95.3  98.9  97.1  4.9  87.0
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation reached 5 %. Both replicates of the test item showed very good correspondence.
Degradation values of more than 100 % were calculated. This is due to the fact that the measurement errors of test flasks and control flasks add up when using the equation proposed in the guideline.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 84.3 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 181.4 mg/L”.
For pure substances, ready biodegradability is defined in the guidelines as degradation surpassing 60 % within 10 days after reaching a level of 10 %.
Because the test item is a mixture, the 10-day window has not to be taken into account.
Therefore, regardless of the 10-day-window the test item is considered as “readily biodegradable within 28 days”.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.
Executive summary:

The test item was tested under GLP conditions and according to OECD guideline 301B and EU Method C.4 -C using a concentration of nominally 20 mg organic carbon/L (corresponding to 181.4 mg test item/L) in test medium.

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days. All validity criteria were met. Degradation of the positive control was 80 % after 9 days.

The following data were determined for the test item :

10-day-window:                                             day 2 – 12

degradation at the end of 10-day-window       95 %

degradation at the end of the test                     97 %

pass level following guideline:                          60 % at the end of 10-day-window for pure substances

respective 60 % at the end of the test for mixtures

Therefore, when applying the 10-day-window, the sample is readily biodegradable following OECD 301B and EU C.4-C respectively.

Because the test item is a mixture, the 10-day-window does not have to be taken into account. Therefore, regardless of the 10-day-window the test item is considered as “readily biodegradable” within 28 days.

Description of key information

The test item was tested under GLP conditions and according to OECD guideline 301B and EU Method C.4 -C using a concentration of nominally 20 mg organic carbon/L (corresponding to 181.4 mg test item/L) in test medium.

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days. All validity criteria were met. Degradation of the positive control was 80 % after 9 days.

The following data were determined for the test item :

10-day-window:                                             day 2 – 12

degradation at the end of 10-day-window       95 %

degradation at the end of the test                     97 %

pass level following guideline:                          60 % at the end of 10-day-window for pure substances

respective 60 % at the end of the test for mixtures

Therefore, when applying the 10-day-window, the sample is readily biodegradable following OECD 301B and EU C.4-C respectively.

Because the test item is a mixture, the 10-day-window does not have to be taken into account. Therefore, regardless of the 10-day-window the test item is considered as “readily biodegradable” within 28 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information