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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:
From September 2017 to January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted July 17, 1992
Deviations:
yes
Remarks:
Deviations are considered to considered to have no effect on the outcome of the study
Qualifier:
according to
Guideline:
other: ISO 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:
yes
Remarks:
Deviations are considered to considered to have no effect on the outcome of the study
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Identification: 1,3-Bis (4-hydroxy benzoyl) benzene
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Pre-treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.13 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (30 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
16 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 CONDITIONS
- Composition of medium: mineral medium as described in the OECD guideline
- Test temperature: 22°C +/- 2 °c
- pH: 7.6
- pH adjusted: yes
- Suspended solids concentration: The concentration of suspended solids was determined to be 4.13 g/L in the concentrated sludge.
- Continuous darkness: yes coloured glass bottles, The test media were excluded from light.
- Test duration : 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the positive and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.
- Test vessels : 2 litre brown coloured glass bottles.
- aeration : 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. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

TEST SYSTEM
- Pre-incubation medium:
The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Type and number of bottles:
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).
- Preparation:
At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle


SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes


STATISTICAL METHODS:
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
5
Sampling time:
28 d
Remarks on result:
other: mean of both duplicate (6% and 4%)
Details on results:
- Theoretical CO2 Production:
The ThCO2 of 1,3-Bis (4-hydroxy benzoyl) benzene was calculated to be 2.76 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

- Biodegradation:
The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of 1,3-Bis (4-hydroxy benzoyl) benzene (6% and 4%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (39%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve. The positive control item was biodegraded by at least 60% (83%) within 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of 1,3-Bis (4-hydroxy benzoyl) benzene (6% and 4%, based on ThCO2).
In the toxicity control, 1,3-Bis (4-hydroxy benzoyl) benzene was found not to inhibit microbial activity.
In conclusion, 1,3-Bis (4-hydroxy benzoyl) benzene was designated as not readily biodegradable.
Executive summary:

The objective of this Carbon dioxide (CO2) evolution test (modified Sturm test) study was to evaluate the non-volatile test item 1,3-Bis (4-hydroxy benzoyl) benzene for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge. The study was in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

1,3-Bis (4-hydroxy benzoyl) benzene was an off-white crystalline powder. The test item was tested in duplicate at a target concentration of 16 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of 1,3-Bis (4-hydroxy benzoyl) benzene was calculated to be 2.76 mg CO2/mg.

The study consisted of six bottles:

             2 inoculum blanks (no test item),

             2 test bottles (1,3-Bis (4-hydroxy benzoyl) benzene),

             1 positive control (sodium acetate) and

             1 toxicity control (1,3-Bis (4-hydroxy benzoyl) benzene plus sodium acetate).

Since 1,3-Bis (4-hydroxy benzoyl) benzene 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. To this end, 10 mL of Milli- RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. 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 no biologically relevant biodegradation of 1,3-Bis (4-hydroxy benzoyl) benzene (6% and 4%, based on ThCO2).

In the toxicity control, 1,3-Bis (4-hydroxy benzoyl) benzene 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,1,3-Bis (4-hydroxy benzoyl) benzene was designated as not readily biodegradable

Description of key information

The ultimate aerobic biodegradability of the test item was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301B and ISO standard 10634. The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of the test item (6% and 4%, based on ThCO2).

The test item is thus considered not "readily biodegradable".

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test item was investigated in one GLP-compliant study performed in accordance with standard methods, with deviations but not impacting the outcome of the study. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.