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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:
2018-03-20 to (experimental phase 2018-04-19 to 2018-05-17)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Lot/batch No.of test material: M-1025-157
- CAS number: 39464-70-5
- Appearance: Clear, yellow-amber liquid
- Expiration date of the lot/batch: 25 October 2018
- Purity: 100%
- Storage conditions: Room temperature (15-25 °C, below 70 % relative humidity (RH))
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant
- Origin: 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 approximately 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.:
4.7 mg/L
Based on:
ThOD
Remarks:
Based on the theoretical oxygen demand (ThOD) of 1.29 mg O2/mg test item, the stock solution was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 4.7 mg/L test item, with a ThOD of about 6.063 mg O2/L).
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 20.6 - 23.2 °C, in the dark. The temperature was measured on weekdays during the experiment. The oxygen concentration of test water was 9.0 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 7.34. The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF TEST SOLUTIONS:
Before the preparation of the respective test solutions with Ethoxylated phenol phosphate an aqueous stock solution of the test item with a concentration of 47 mg/L was prepared and diluted accordingly. During the preparation of the test solutions they were mixed by mechanical stirring using ultrasonic bath for 10 minutes. The test item concentration in the test solutions was 4.7 mg/L. The chosen test item concentration was based on theoretical oxygen demand (ThOD) of 1.29 mg O2/mg test item (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test.
a) Test item:
Based on the theoretical oxygen demand (ThOD) of 1.29 mg O2/mg test item, test item stock solution (47 mg/L) (corresponding to 15.98 mg of Ethoxylated phenol phosphate) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 4.7 mg/L test item, with a ThOD of about 6.063 mg O2/L).
b) Procedure Control: Sodium benzoate:
Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution (360 mg/L) corresponding to 12.168 mg of Sodium benzoate was mixed into 3.38 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
c) Inoculum Control:
Only filtered inoculum was added to 3.38 litres of aqueous test medium.
d) Toxicity Control:
The test item (340 mL) and the reference item stock solutions (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 4.7 mg/L test item (ThOD of 6.063 mg O2/L) and 3.6 mg/L reference item (ThOD 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.
Test Bottles:
The number of test bottles was the follow:
- 10 bottles containing the test item and inoculum
- 10 bottles containing the reference item and inoculum (procedure control)
- 10 bottles containing only inoculum (inoculum control)
- 10 bottles containing the test item, reference item and inoculum (toxicity control)


MEASUREMENTS:
- 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.
- Temperature:
Temperature was measured continuously and registered on weekdays.


Reference substance:
other: sodium benzoate
Key result
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Ethoxylated phenol phosphate reached a mean of 5.0 % after 28 days based on the ThOD of the test item.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 70.0 % after 14 days, and to a mean of 74.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

Biodegradation of the Toxicity control:

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 37.9 % biodegradation was noted within 14 days and 40.4 % 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 4.7 mg/Lon the secondary effluent microorganisms because degradation was >25 % within 14 days.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions the percentage biodegradation of Ethoxylated phenol phosphate reached a mean of 5.0 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.
Executive summary:

The test item Ethoxylated phenol phosphate was investigated for its ready biodegradability in a Closed Bottle Test conducted according to OECD Guideline 301D. Under the test conditions the percentage biodegradation of  Ethoxylated phenol phosphate  reached a mean of 5.0 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable. The reference item, Sodium benzoate, was sufficiently degraded to a mean of  70.0 % after 14 days, and to a mean of  74.2 % after 28 days of incubation, based on ThOD, 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 37.9 % biodegradation was noted within 14 days and 40.4 % 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 4.7 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

Description of key information

Under the test conditions the percentage biodegradation of Ethoxylated phenol phosphate reached a mean of 5.0 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.

Key value for chemical safety assessment

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

Additional information

The test item Ethoxylated phenol phosphate was investigated for its ready biodegradability in a Closed Bottle Test conducted according to OECD Guideline 301D.

Under the test conditions the percentage biodegradation of  Ethoxylated phenol phosphate  reached a mean of 5.0 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.Therefore, the test item is considered not readily biodegradable.The reference item, Sodium benzoate, was sufficiently degraded to a mean of  70.0 % after 14 days, and to a mean of  74.2 % after 28 days of incubation, based on ThOD, 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 37.9 % biodegradation was noted within 14 days and 40.4 % 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 4.7 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.