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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:
25 February 2019 - 04 April 2019
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 July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
other: SO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
SOURCE
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.

TREATMENT
Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 3.4 g/L in concentrated
sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 10 mg/L.

REASON FOR SELECTION
The test has been accepted internationally for determining 'ready' biodegradability of test items under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
17 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST PROCEDURE AND CONDITIONS

TEST DURATION
28 days for inoculum blank and test item (last CO2 measurement on day 29).
14 days for procedure and toxicity control (last CO2 measurement on day 15).
During the test period, test media were aerated and stirred continuously.
TEST VESSELS
2 litre brown coloured glass bottles.
MILLI- RO WATER
Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
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 Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
(B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
(C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
(D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
MINERAL MEDIUM
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

BARIUM HYDROXIDE
0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.
SYNTHETIC AIR (CO2 < 1 PPM)
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
ILLUMINATION
Test media were excluded from light.

SAMPLING AND CONTROLS

Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for inoculum blank and test item. Titrations for procedure and toxicity control were made over a period of at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle.
A new CO2-absorber was placed at the far end of the series.
Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the inoculum blank and test suspension.
Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).

The study consisted of six bottles:
2 inoculum blanks (no test item),
2 test bottles (Esacure 3644),
1 procedure control (sodium acetate) and
1 toxicity control (Esacure 3644 plus sodium acetate).

Reference substance:
acetic acid, sodium salt
Test performance:
Since all criteria for acceptability of the test were met, this study was considered to be valid.
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Since Esacure 3644 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Furthermore, test medium was swirled around daily, since the test item tended to float on the water surface. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).
Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of Esacure 3644 (2% and 0%, based on ThCO2).
In the toxicity control, Esacure 3644 was found not to inhibit microbial activity.
Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Esacure 3644 was designated as not readily biodegradable.
Results with reference substance:
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve

For full results see appendix 1 attached to background information

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In conclusion, Esacure 3644 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the study was to evaluate test item Esacure 3644 for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

Procedures described in this report were in compliance with OECD guideline No. 301 B, 1992. In addition, procedures were designed to meet test methods of ISO standard 10634, 1995.  

Esacure 3644 was a light yellow solid (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. Total Organic Carbon (TOC) content of the test item was determined to be 71.54%. Based on TOC content ThCO2 of the test item was calculated to be 2.62 mg CO2/mg. The test item was tested in duplicate at a target concentration of 17 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles:  

2 inoculum blanks (no test item),

2 test bottles (Esacure 3644),

1 procedure control (sodium acetate) and

1 toxicity control (Esacure 3644 plus sodium acetate).

Since Esacure 3644 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Furthermore, test medium was swirled around daily, since the test item tended to float on the water surface. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of Esacure 3644 (2% and 0%, based on ThCO2).

In the toxicity control, Esacure 3644 was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Esacure 3644 was designated as not readily biodegradable.

Description of key information

Study conducted to recognised testing guidelines with GLP certification

Key value for chemical safety assessment

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

Additional information