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Biodegradation in water: screening tests

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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:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7703200
- Expiration date of the lot/batch: 2019-02-28
- 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.:
2.47 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
- The respective amount of Phosphoric acid, C8-12 (even numbered) alkyl esters was weighed directly to reach the required test item concentration of 2.47 mg/L.
- The chosen test item concentration was based on the measured chemical oxygen demand (COD): 2.44 ±0.031 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 2.44 mg O2/mg test item, 8.4 mg of Phosphoric acid, C8-12 (even numbered) alkyl esters was thoroughly mixed into 3.40 litres of aqueous test medium1 (corresponding to 2.47 mg/L test item, with a COD of about 6.027 mg O2/L).
1During the preparation of the test solution ultrasonic bath was used.
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 (8.4 mg)1 and reference item stock solution* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 2.47 mg/L test item (COD of 6.027 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).
* The concentration of the reference item stock solution was: 360 mg/L.
1During the preparation of the test solution ultrasonic bath was used.

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:
65.5
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
56.4
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
53.3
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
41.5
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 (2.47 mg/L test item; 3.6 mg/L reference substance): 26.9 % biodegradation after 7 days, 44.5 % biodegradation after 14 days, 47.7 % biodegradation after 21 days, 51.8 % biodegradation after 28 days.

Please refer to tables attached.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results of this study, the test item is considered readily biodegradable.
Executive summary:

The test item Phosphoric acid, C8-12 (even numbered) alkyl esters 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 Phosphoric acid, C8-12 (even numbered) alkyl esters reached a mean of 65.5 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is 60 % of ThOD and this level has to be reached in a 10-day window within the 28-day period of the test, or if the test item is sufficiently degraded to above 60 % after 14 days the value obtained in a 14-day window is acceptable in the used method. Taking into account that none of these criteria was met during the study, the test item should be considered not readily biodegradable. Nevertheless, according the CSTEE (Opinion of the Scientific Committee on Toxicity, Ecotoxicity and the Enviroment), the 10 days window criterion is not a requirement for the desired stringency in the particular case of surfactants and it is not necessary to keep the "10 days window" for assessing ready ultimate biodegradability of surfactants in detergents. Taking into account that the test item is a surfactant and its percentage biodegradation reached a mean of 65.5 % after 28 days, therefore the test item is considered readily biodegradable. 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 44.5 % biodegradation was noted within 14 days and 51.8% 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 2.47 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 readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar ecotoxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, C8-12 (even-numbered) alkyl esters [EC not yet assigned; CAS not assigned]
Target: Phosphoric Acid, linear and branched Octyl Esters [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Both the Source and Target substances are alkyl phosphate esters with an alkyl chainlength of either C8 (target) or C8-12 (source). It is not expected that branching will have an effect on ready biodegradability as shown by data available for other phosphate esters. Therefore, it is considered to be justified to read across from teh surce to the target.
4. DATA MATRIX
Source: Readily biodegradable
Target: No data
Reason / purpose:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
65.5
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
56.4
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
53.3
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
41.5
Sampling time:
7 d

Description of key information

The substance is ready biodegradable, reaching 65.5 % biodegradation after 28 days (read-across Phosphoric acid, C8 -12 (even-numbered) alkyl esters.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The test item Phosphoric acid, C8-12 (even numbered) alkyl esters 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 Phosphoric acid, C8-12 (even numbered) alkyl esters reached a mean of 65.5 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is 60 % of ThOD and this level has to be reached in a 10-day window within the 28-day period of the test, or if the test item is sufficiently degraded to above 60 % after 14 days the value obtained in a 14-day window is acceptable in the used method. Taking into account that none of these criteria was met during the study, the test item should be considered not readily biodegradable. Nevertheless, according the CSTEE (Opinion of the Scientific Committee on Toxicity, Ecotoxicity and the Enviroment), the 10 days window criterion is not a requirement for the desired stringency in the particular case of surfactants and it is not necessary to keep the "10 days window" for assessing ready ultimate biodegradability of surfactants in detergents. Taking into account that the test item is a surfactant and its percentage biodegradation reached a mean of 65.5 % after 28 days, therefore the test item is considered readily biodegradable. 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 44.5 % biodegradation was noted within 14 days and 51.8% 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 2.47 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 readily biodegradable.