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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:
09-01-2018 to 22-03-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Laboratory culture: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
33 mg/L
Based on:
test mat.
Remarks:
corresponding to 12 mg TOC/L; target concentration
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 – 23 °C. See table 2.
- pH: Measured prior to and completion of test. See table 2.
- pH adjusted: No
- Aeration of dilution water: The freshly obtained sludge was kept under continuous aeration until further treatment. During the test period, the test media were aerated and stirred continuously. On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension.
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2-litres test bottles
- Number of culture flasks/concentration: In duplicate. Test suspension: containing test item and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Positive control: containing reference item and inoculum (1 bottle). Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Method used to create aerobic conditions: Not reported
- Measuring equipment: The respirometer used during this study is an Oxitop Control System. The consumption of oxygen is determined by measuring the pressure drop in the respirometer flask. Evolved carbon dioxide is absorbed in sodium hydroxide pellets.

SAMPLING
- Sampling frequency: Every second or third day.
- Sampling method: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L
Test performance:
The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item. Therefore, the test is considered valid.
Parameter:
% degradation (CO2 evolution)
Remarks:
mean (n = 2)
Value:
77
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Remarks:
replicate 1
Value:
82
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Remarks:
replicate 2
Value:
72
Sampling time:
28 d
Details on results:
The test item undergoes mean relative biodegradation of 77% after 28 days (82% and 72% in replicates in the test conditions. The 10-day window criterion need not be applied.
Results with reference substance:
Degradation of sodium acetate sale exceeded 60% after 14 days (actual 90%): the activity of the inoculum was thus verified (validity criterion).

Acceptability criteria:

1. The reference item was biodegraded by at least 60% (actual: 90%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20 (actual: ≤ 10%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (61.5 mg CO2 per 2 litres of medium, corresponding to 30.8 mg CO2/L).

4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

All acceptability criteria were considered to have been met.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of this study, the test item undergoes 72 to 82% biodegradation after 28 days based on CO2 evolution (considering the ThCO2). Since the test item is a UVCB the 10-day window criterion need not be applied. The test item is considered to be readily biodegradable.
Executive summary:

The ready biodegradability test was carried out according to OECD TG 301B guideline under GLP. The test item was a yellow liquid UVCB and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test item was determined to be 36.23%. Based on the TOC content the ThCO2 of the test item was calculated to be 1.33 mg CO2/mg. The test item was tested in duplicate at a target concentration of 33 mg/L, corresponding to 12 mg TOC/L. Handling of the test item was performed under low light conditions. The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant receiving predominantly domestic sewage. The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L. The study consisted of six bottles: two inoculum blanks (no test item), two test bottles (test item), one positive control (sodium acetate) and one toxicity control (test item plus sodium acetate). The test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15). The relative biodegradation values calculated from the measurements performed during the test period revealed 82% and 72% biodegradation of the test item, for vessel A and B, respectively (based on ThCO2). All relevant acceptability criteria were met. Under the conditions of this study, the test item undergoes 72 to 82% biodegradation after 28 days based on CO2 evolution. Since the test item is a UVCB the 10-day window criterion need not be applied. The test item is considered to be readily biodegradable.

Description of key information

Biodegradation: readily biodegradable, mean biodegradation 77% (28-days). 10 -day window not applicable to multi-constituent/UVCB substances consisting of structurally similar constituents (OECD, 2006); OECD TG 301B, 2018

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study : OECD TG 301B, 2018 : The ready biodegradability test was carried out according to OECD TG 301B guideline under GLP. The test item was a yellow liquid UVCB and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test item was determined to be 36.23%. Based on the TOC content the ThCO2 of the test item was calculated to be 1.33 mg CO2/mg. The test item was tested in duplicate at a target concentration of 33 mg/L, corresponding to 12 mg TOC/L. Handling of the test item was performed under low light conditions. The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant receiving predominantly domestic sewage. The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L. The study consisted of six bottles: two inoculum blanks (no test item), two test bottles (test item), one positive control (sodium acetate) and one toxicity control (test item plus sodium acetate). The test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15). The relative biodegradation values calculated from the measurements performed during the test period revealed 82% and 72% biodegradation of the test item, for vessel A and B, respectively (based on ThCO2). All relevant acceptability criteria were met. Under the conditions of this study, the test item undergoes 72 to 82% biodegradation after 28 days based on CO2 evolution. Since the test item is a UVCB the 10-day window criterion need not be applied. The test item is considered to be readily biodegradable.

 

Applicant assessment indicates: The test item was biodegraded by 77% at day 28 in the CO2 evolution Test. The complex multi-constituent (UVCB) test item is therefore classified as readily biodegradable. The 10-day window that can be applied to CO2 Evolution, where 60% biodegradation must be achieved following the attainment of 10 % biodegradation, is not applied to multi-constituent or UVCB substances consisting of structurally similar constituents (OECD, 2006).

 

References:

1. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7b: Endpoint Specific Guidance, R.7.9.4, June 2017)

2. Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation, Section 3 (OECD, September 2006)

3. GHS revision 4 (2011), Globally harmonized system of classification and labelling of chemicals (GHS). Chapter 4.1. Hazardous to the aquatic Environment. United Nations, 2011

4. ECHA Guidance on Application on the CLP Criteria, section 4.1.3.2.3.2 (v5.0, July 2017)

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