Phosphoric acid, mono and bis-linear butyl esters, potassium salts

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:

21st July - 18th August, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7675229
- Expiration date of the lot/batch: 2018-08-31
- Storage condition: Controlled room temperature (15-25°C, below 70 RH%)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Secondary effluent, microorganisms from a domestic waste water treatment plant. The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
- Conditioning: The secondary effluent used for this study was allowed to settle for an hour, then the decanted effluent was aerated until use (not later than an hour).

Duration of test (contact time):

28 d

Initial conc.:

5.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST UNITS
- Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
- Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.

TEST CONDITIONS
- The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2°C according to guideline. The test flasks were placed into an incubator and kept at 21.0 - 23.4°C, in the dark. The temperature was measured on weekdays, but at least each day of oxygen consumption measurement during the experiment.
- The oxygen concentration of test water was 8.4 mg/L at the start of the test.
- The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.25.

PREPARATION OF THE TEST SOLUTIONS
- Before the preparation of the respective test solutions with Phosphoric acid, butyl ester, potassium salt and aqueous stock solution of the test item with a concentration of 53 mg/L was prepared and diluted accordingly. During the preparation of the test solutions they were mixed by mechanical stirring and using ultrasonic bath (5 minutes) to ensure a good dispersion. The test item concentration in the test solutions was 5.3 mg/L.
- The chosen test item concentration was based on the measured chemical oxygen demand (COD): 1.13 ±0.029 mg O2/mg test item and on the performed 14-day preliminary test.
- The components were applied in the amounts/volumes in the test flasks:
1.) Test Item (flasks 1a and 1b)
Based on the measured chemical oxygen demand (COD) of 1.13 mg O2/mg test item, test item stock solution (corresponding to 17.914 mg of Phosphoric acid, butyl ester, potassium salt) was thoroughly mixed into 3.38 litres of aqueous test medium (corresponding to 5.3 mg/L test item, with a COD of about 5.9897 mg O2/L).
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.240 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).
* The concentration of the stock solution was: 360 mg/L.
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.40 litres of aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b)
The Test Item stock solution (338 mL) and reference item stock solution (33.8 mL) were mixed into 3.38 litres of aqueous test medium corresponding to 5.3 mg/L test item (COD of 5.989 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

COURSE OF THE TEST
- Preparation of Test Flasks: Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well
to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.
- The Test Bottles: 10 bottles per test set were used

MEASUREMENTS
- COD Measurement: The COD (chemical oxygen demand) of the test item was determined in the analytical department of the test facility using Lovibond® COD Measuring System.
- Measurement of Oxygen: The incubation period of the closed bottle test was 28 days. The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

2.5

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

3.3

Sampling time:

7 d

Results with reference substance:

Reference substance (3.6 mg/L): 66.7 % biodegradation after 7 days, 79.2 % biodegradation after 14 days, 81.7 % biodegradation after 21 days, 81.7 % biodegradation after 28 days.
Toxicity control (5.3 mg/L test item; 3.6 mg/L reference substance): 32.9 % biodegradation after 7 days, 41.0 % biodegradation after 14 days, 41.8 % biodegradation after 21 days, 43.8 % biodegradation after 28 days.

Please refer to tables attached.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Based on the results of this study, the test item is considered not readily biodegradable.

Executive summary:

The test item was investigated for its ready biodegradability in a Closed Bottle 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 item, and functioned as a procedure control. The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use. Under the test conditions the percentage biodegradation of the test item reached a mean of 5.0 % after 28 days based on the measured COD of the test item. Therefore the test item is considered not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. The reference item Sodium benzoate was sufficiently degraded to a mean of 79.2% after 14 days, and to a mean of 81.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum. In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 41.0 % biodegradation was noted within 14 days and 43.8% biodegradation after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 5.3 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days. The study met the validity criteria. Based on the results of this study, the test item is considered not readily biodegradable.

Description of key information

The test item is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The test item was investigated for its ready biodegradability in a Closed Bottle 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 item, and functioned as a procedure control. The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use. Under the test conditions the percentage biodegradation of the test item reached a mean of 5.0 % after 28 days based on the measured COD of the test item. Therefore the test item is considered not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. The reference item Sodium benzoate was sufficiently degraded to a mean of 79.2% after 14 days, and to a mean of 81.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum. In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 41.0 % biodegradation was noted within 14 days and 43.8% biodegradation after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 5.3 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days. The study met the validity criteria. Based on the results of this study, the test item is considered not readily biodegradable.