Tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate

Biodegradation in water: screening tests

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:

2018-01-15 - 2018-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO standard 10634

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, 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
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.:

12 mg/L

Based on:

TOC

Initial conc.:

16.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of test solutions
Since the test substance 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 (test item bottle A: 32.62 mg; test item bottle B: 32.60 mg and toxicity control bottle: 32.73 mg). 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 the test organisms.
Any residual volumes were discarded.

Testing strategy and experimental design
Test conditions
pH
At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.

Temperature of medium
Continuously in a vessel with Milli- RO water in the same room.

Preparation of Bottles
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.

Test Procedure and Conditions
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.

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 andmade up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water andmade 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 the 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. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination
The test media were excluded from light.

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

13

Sampling time:

28 d

Remarks on result:

other: Bottle A

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Bottle B

Details on results:

Between nominal day 24 and 25 there was a period without aeration because of clogged tubing.
Evaluation: Such a short period without aeration has no impact on the validity or results of the study.

Results with reference substance:

Reference item sodium acetate showed a normal biodegradation curve.

Theoretical CO₂Production

The ThCO₂ of test-substance was calculated to be 2.63 mg CO₂/mg.

The ThCO₂ of sodium acetate was calculated to be 1.07 mg CO₂/mg.

Biodegradation

Attached figure shows the curves for biodegradation of the two bottles with the test substance, the positive control and the toxicity control.

The relative biodegradation values calculated from the measurements performed during the test period revealed 13% and 0% biodegradation of test-substance (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (41%, 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.

Monitoring of Temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C.

pH Values

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.7 → 7.6^1	-	7.5
Blank control (B)	7.7 → 7.6^1	-	7.5
Positive control	7.7 → 7.6^1	7.7	-
test-substance (A)	7.6	-	7.6
test-substance (B)	7.6	-	7.6
Toxicity control	7.6	7.7	-

¹: Adjusted using 1 M HCl

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In conclusion, test substance was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

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

The study procedures described in this report were 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. The study was conducted according to GLP. The test item was tested in duplicate at a target concentration of 16.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO₂production (ThCO₂) of test-substance was calculated to be 2.63 mg CO₂/mg.

The study consisted of six bottles:

·        2 inoculum blanks (no test item),

·        2 test bottles (Lexolube1 - Pyromellitate),

·        1 positive control (sodium acetate) and

·        1 toxicity control (Lexolube1 - Pyromellitate plus sodium acetate).

Since the sample 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. Test duration was 28 days for the inoculum blank and test item (last CO₂measurement on day 29) and 14 days for the positive and toxicity control (last CO₂measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 13% and 0% biodegradation of test item (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, test sample 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, the test substance was designated as not readily biodegradable.

Description of key information

Determination of 'Ready' Biodegradability was performed in a Carbon Dioxide (CO₂) Evolution Test (Modified Sturm Test) 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. The study was conducted according to GLP. The test item was tested in duplicate at a target concentration of 16.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO₂production (ThCO₂) of test-substance was calculated to be 2.63 mg CO₂/mg. The relative biodegradation values calculated from the measurements performed during the test period revealed 13% and 0% biodegradation of test item (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10 -day window) was not met. In conclusion, test substance was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Key information:

The objective of the key study (Timmer, 2018) was to evaluate the test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

The study procedures described in this report were 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. The study was conducted according to GLP.

Test item was a clear colourless liquid with a purity of 99.8%. The test item was tested in duplicate at a target concentration of 16.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO₂production (ThCO₂) of test-substance was calculated to be 2.63 mg CO₂/mg.

The study consisted of six bottles:

·        2 inoculum blanks (no test item),

·        2 test bottles (Lexolube1 - Pyromellitate),

·        1 positive control (sodium acetate) and

·        1 toxicity control (Lexolube1 - Pyromellitate plus sodium acetate).

Since the sample 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. Test duration was 28 days for the inoculum blank and test item (last CO₂measurement on day 29) and 14 days for the positive and toxicity control (last CO₂measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 13% and 0% biodegradation of test item (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, test sample 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, the test-substance was designated as not readily biodegradable.

Supporting information:

Ready Biodegradability Sarpy rule set by Istituto di Ricerche Farmacologiche Mario Negri from VEGA QSAR Model:

Prediction of tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate is NON Readily Biodegradable using the model.

The substance is not readily biodegradable.

BIOWIN 1 and 2 models by EPI Suite software v4.1.

Prediction is "biodegrades fast".

The substance is expected to biodegrade fast.

Biowin 3 from EPI Suite v4.1 software: the expected ultimate biodegradation timeframe is days to weeks

Biowin 4 from EPI Suite v4.1 software: the expected primary biodegradation timeframe is days

Biowin 5 from EPI Suite v4.1 software: the substance is expected to be readily biodegradable.

Biowin 6 from EPI Suite v4.1 software: the substance is expected to be readily biodegradable.

The test substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate is expected to biodegrade fast.