ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

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:

05 September 2019 to 03 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Method of conditioning: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This was used for the experiment.
- Concentration of sludge: Final sludge concentration in test flasks: 28.7 mg sludge/L

Duration of test (contact time):

28 d

Initial conc.:

102.5 mg/L

Based on:

test mat.

Remarks:

(corresponding to an oxygen demand of about 238.1 mg/L (ThODNH4))

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water, composed of the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1 000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume.
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume.
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume.
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
- pH adjusted: Yes. The pH was adjusted to pH 7.5 with 1M HCl solution.
- Additional substrate: No
- Solubilising agent: No, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test material.
- Test temperature: 22 ± 1 °C
- pH: 7.1 - 7.6. At the beginning of the test the pH value of the test material was 7.6.
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Measuring equipment: BSB/BOD-Sensor-System
- Preparation of test flasks: The test water was added via measuring cylinder and the activated sludge was added via Eppendorf pipette to the test flasks. The amounts of test material and reference material were directly weighed into the test flasks. No emulsifiers or solvents were used but the solutions were dispersed by continuous stirring (via magnetic stirring plats) to achieve a homogeneous solution of the test material.
- Incubation: The closed test flasks were incubated in a climatised chamber under continuous stirring (via magnetic stirring plates). The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved CO2 was absorbed in an aqueous solution (45 %) of potassium hydroxide.
The change of pressure in the reaction vessels was measured by means of the BSB/BOD-Sensor-System.
The temperature was recorded by means of the automated software AMR Wincontrol©.

CONTROL AND BLANK SYSTEM
- The study included 2 replicates of treatment with test material, 2 inoculum control replicates. Single determinations were made for the procedure control, abiotic control and toxicity control.

RESULT EVELUATION
The percentage biodegradation of the test material and of the reference item sodium benzoate was calculated according to the following formula:

% degradation = (BODControl adjusted [mg/L] / ThOD [mg O2/L] ) x 100

where:
BODControl adjusted = BOD of test material (or reference item) – mean BOD of inoculum controls
ThOD [mg O2/L] = (Test material or reference item [mg] * ThOD [mg O2/mg test material or reference item] ) / 0.244 L

For the toxicity control, the ThOD is the sum of the ThOD of the test material and the ThOD of the reference item weighed in the toxicity control flask.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (DOC removal)

Value:

6

Sampling time:

28 d

Details on results:

- Percentage biodegradation
The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test material of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test material reaches at least 10 % degradation.
Under the conditions of this study, the test material never reached 10 % biodegradation. The mean biodegradation percentage at the end of the 28-day exposure period was 6 % (ThODNH4).

In the toxicity control, both the test material and the reference material, sodium benzoate, 30 % (ThODNH4) biodegradation was noted within 14 days and 31 % (ThODNH4) biodegradation after 28 days of incubation. According to the test guidelines the test can be assumed not to be inhibitory to the aerobic activated sludge microorganisms because degradation was > 25 % within 14 days.

The oxygen demand in the abiotic control was 0 mg/L during the test duration. Therefore, there was no need to correct the degradation of the test material and toxicity control.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 75 % after 14 days and to 78 % after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Validity Criteria of the Study

- Inoculum control: The oxygen demand of the inoculum control (medium and inoculum) was 2.5 mg/L O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

- pH-value: The pH of the test material flasks at the end of the test was 7.4 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

- Reference material: The percentage degradation of the reference material should reach the level for ready biodegradability (> 60 %) within 14 days as required by the test guideline. The reference material, sodium benzoate, was degraded to more than 60 % after 4 days of incubation.

- Test material: The difference of duplicate values for the degradation of the test material at the plateau at the end of the test and at the end of the ten-day window was less than 20 %. The difference of duplicate values at day 28 differed by 2 %.

The validity criterion was fulfilled.

- Toxicity control: If in a toxicity test, containing both the test material and a reference material less than 25 % biodegradation (based on ThODNH4) occurred within 14 days, the test material can be assumed to be inhibitory. The biodegradation was 30 % at day 14. The test material was no inhibitory.

Cumulative Biochemical Oxygen Demand (mgO2/L) in Test Flasks During the Test Period of 28 Days.

Time (Days)	Flask No.
	1	2	3	4	5	6	7
1	0	0	0	0	30	0	35
2	5	5	0	0	70	0	75
3	5	5	0	5	95	0	90
4	5	5	0	5	105	0	105
5	5	5	0	5	110	0	110
6	5	5	0	5	15	0	115
7	5	5	0	5	120	0	120
8	5	10	0	5	120	0	120
9	5	10	0	5	125	0	125
10	5	15	0	5	125	0	125
11	5	15	0	5	125	0	125
12	5	15	0	5	125	0	125
13	5	15	0	5	130	0	125
14	5	15	0	5	130	0	125
15	5	15	0	5	130	0	125
16	5	15	0	5	130	0	125
17	5	15	0	5	135	0	125
18	5	15	0	5	135	0	125
19	5	15	0	5	135	0	130
20	10	15	0	5	135	0	130
21	10	15	0	5	135	0	130
22	10	20	0	5	135	0	130
23	10	20	0	5	135	0	130
24	10	20	0	5	135	0	130
25	10	20	0	5	135	0	130
26	10	20	0	5	135	0	130
27	10	20	0	5	135	0	130
28	15	20	0	5	135	0	130

Flasks 1 & 2: Test material.

Flasks 3 & 4: Inoculum control.

Flask 5: Reference (procedure control).

Flask 6: Abiotic control.

Flask 7: Toxicity control.

 

Percentage Biodegradation of the Test Material, of Sodium Benzoate and of the Toxicity Control Based on ThODNH4

Time (Days)	Percentage Biodegradation
	Test material*			Sodium Benzoate**	Toxicity Control
	Flask 1 [%]	Flask 2 [%]	Mean [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	0	18	9
2	2	2	2	41	18
3	1	1	1	54	21
4	1	1	1	60	25
5	1	1	1	63	6
6	1	1	1	66	27
7	1	1	1	69	29
8	1	3	2	69	29
9	1	3	2	72	30
10	1	5	3	72	30
11	1	5	3	72	30
12	1	5	3	72	30
13	1	5	3	75	30
14	1	5	3	75	30
15	1	5	3	75	30
16	1	5	3	75	30
17	1	5	3	78	30
18	1	5	3	78	30
19	1	5	3	78	31
20	3	5	4	78	31
21	3	5	4	78	31
22	3	7	5	78	31
23	3	7	5	78	31
24	3	7	5	78	31
25	3	7	5	78	31
26	3	7	5	78	31
27	3	7	5	78	31
28	5	7	6	78	31

* ThODNH4 of the test material: 2.324 mg O2/mg test material.

** ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference material.

 

pH Values at the End of the Test

Flask No.	Treatment	pH Value
1	Test material	7.4
2	Test material	7.4
3	Inoculum control	7.6
4	Inoculum control	7.5
5	Reference material (Procedure control)	7.1

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of the study the mean biodegradation of the test material was 6 % (ThODNH4) after 28 days. The degradation rate of the test material never reached 60 % within the 10-day window and after 28 days. Therefore, the test material is considered to be not readily biodegradable.

Executive summary:

The ready biodegradablity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines EU Method C.4-D and OECD 301F, under GLP conditions.

The study utilised aerobic activated sludge from a domestic wastewater treatment plant. During the study, the test material was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control. Degradation rate of test material was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.

The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test material of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test material reaches at least 10 % degradation.

Under the conditions of the study the mean biodegradation of the test material was 6 % (ThODNH4) after 28 days. The degradation rate of the test material never reached 60 % within the 10-day window and after 28 days. Therefore, the test material is considered to be not readily biodegradable.

Description of key information

The ready biodegradablity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines EU Method C.4-D and OECD 301F, under GLP conditions.

The study utilised aerobic activated sludge from a domestic wastewater treatment plant. Under the conditions of the study the mean biodegradation of the test material was 6 % (ThODNH4) after 28 days. Therefore, the test material is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater