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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:
03 Jul 2017 - 04 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Molecular formula: C13H22O4 (used for calculating ThOD)
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent, domestic, non-adapted
Details on inoculum:
- Source: The source of test organisms was secondary effluent freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: Secondary effluent was filtered through a coarse filter paper; the first 200 mL was discarded. The filtrate was kept aerated until inoculation.
- Inoculation: 4 mL filtrate of secondary effluent per litre of final volume
Duration of test (contact time):
28 d
Initial conc.:
1 mg/L
Based on:
test mat.
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 1 mL of solution (A) to (D) and Milli-RO water (tap-water purified by reverse osmosis)
Stock solutions of mineral components:
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in 1 litre Milli-RO water; pH: 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in 1 litre Milli-RO water.
C) 36.40 g CaCl2.2H2O dissolved in 1 litre Milli-RO water.
D) 0.25 g FeCl3.6H2O dissolved in 1 litre Milli-RO water.
- Test temperature: between 22.0 and 23.1°C.
- pH at t=0 d (blanks): 7.5
- pH at t=0 d (other bottles): 7.4
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 250-300 mL BOD bottles with glass stoppers
- Test set up/number and groups of test bottles:
Test suspension (1 mg/L): containing test substance and inoculum
Test suspension (3 mg/L): containing test substance and inoculum
Inoculum blank: containing only inoculum
Positive control: containing reference substance (2 mg/L) and inoculum
Toxicity control: containing test substance (1 mg/L), reference substance (2 mg/L) and inoculum
Individual BOD bottles were prepared for each measuring point, except that the bottles measured at the start were also measured on day 7.
Parallel groups were prepared to allow duplicate measurements of oxygen consumption at the test intervals.
- Determination of oxygen concentration: In duplicate; immediately at the start of the experiment (day 0) and at day 7, 14, 21 and 28, using WTW oxygen meter supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G.

PREPARATION OF TEST SOLUTIONS
Preparation of the test media started with a clear and colourless stock solution of 10 mg/L by adding 20.3 mg test item to 2000 mL of mineral medium. Magnetic stirring (27 min) was used to accelerate dissolution of the test item in mineral medium. Exact volumes of the stock solution corresponding to the test concentrations (1 and 3 mg/L) and microbial organisms (4 mL/L) were added to the test medium.
Reference substance:
acetic acid, sodium salt
Remarks:
at 2 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
53
Sampling time:
7 d
Remarks on result:
other: at 1 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
14 d
Remarks on result:
other: at 1 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
50
Sampling time:
7 d
Remarks on result:
other: at 3 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
14 d
Remarks on result:
other: at 3 mg/L
Details on results:
The ThOD of Dibutyl itaconate was calculated to be 2.18 mg O2 per mg.
The ThOD of sodium acetate (reference item) was calculated to be 0.78 mg O2/mg.

The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed significant biodegradation of Dibutyl itaconate. At day 7, 14, 21 and 28, respectively:
At 1 mg/L: 53%, 63%, 61% and 52%
At 3 mg/L: 50%, 60%, 67% and 55%
Since biodegradation of at least 60% was reached within 14 days of biodegradation exceeding 10%, Dibutyl itaconate was concluded to be readily biodegradable.
Results with reference substance:
The reference substance was degraded by 70% after 14 days.

In the toxicity control more than 25% biodegradation occurred within 14 days (65%, based on ThOD). Thus, the toxicity control showed that Dibutyl itaconate did not inhibit microbial activity.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the closed bottle test the substance was determined to be readily biodegradable.
Executive summary:

The ready biodegradation of the substance under the conditions of the closed bottle test was investigated according to OECD guideline 301 D and GLP principles. Test concentrations of 1 and 3 mg/L were tested in duplicate during 28 days. The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed significant biodegradation of the substance. At day 7, 53% and 50% biodegradation of the substance at 1 and 3 mg/L, respectively, which was 63% and 60% at day 14. Since biodegradation of at least 60% was reached within 14 days of biodegradation exceeding 10%, the substance was concluded to be readily biodegradable. In the toxicity control, the substance was found not to inhibit microbial activity. The criteria for acceptability of the test were met and the study is considered to be reliable without restriction. In conclusion, the substance was determined to be readily biodegradable under the conditions of the closed bottle test.

Description of key information

Based on the results obtained in a study performed in accordance with OECD 301D (closed bottle test), the substance is designated as readily biodegradable. This conclusion is supported by BIOWIN calculations.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradation of the substance was investigated in two OECD/EC studies. The ready biodegradation of the substance under the conditions of the closed bottle test was investigated according to OECD 301D and GLP principles (reliable without restriction). Test concentrations of 1 and 3 mg/L were tested in duplicate during 28 days. The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed significant biodegradation of the substance. At day 7, 53% and 50% biodegradation of the substance at 1 and 3 mg/L, respectively, which was 63% and 60% at day 14. Since biodegradation of at least 60% was reached within 14 days of biodegradation exceeding 10%, the substance was concluded to be readily biodegradable. In the toxicity control, the substance was found not to inhibit microbial activity. The criteria for acceptability of the test were met and the study is considered to be reliable without restriction. In conclusion, the substance was determined to be readily biodegradable under the conditions of the closed bottle test.

Earlier the ready biodegradation of the substance was investigated according to OECD 301B (modified Sturm test) and GLP principles (reliable with restrictions, as not all validity criteria were met). In this study, the substance was also biodegraded significantly during the test period of 28 days. Only, as the pass level of 60% for ready biodegradability within the 10-day window was not met, it was not designated as readily biodegradable under the conditions of the modified Sturm test.

In accordance with the ECHA Guidance (Chapter R.7b), positive test results should generally supersede negative test results due to the stringent test conditions in the ready biodegradability tests, unless there are strong (structural) reasons to question the positive test results.

The substance is an ester that may hydrolyze in water to form butanol. Butanol is volatile and is expected to disappear in the modified Sturm test, while it does not disappear in the closed bottle test, in which all substances that arise are forced to stay into solution. Based on this and as there is no reason to question the test results obtained with the closed bottle test, the substance is concluded to be readily biodegradable. This conclusion is supported by BIOWIN calculations.