Barium(2+) 12-hydroxyoctadecanoate

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:

from 2017-10-10 to 2018-04-05

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)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

440/2008/EC, 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 03 October 2017 (seven days before the main test).
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. 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 5 g dry material per litre was mixed with test water (mineral medium, see above) and then aerated under test conditions (for 7 days) until use.
- Pretreatment: Pre-conditioning (03-10 October 2017) consisted of aerating (2 L/minute) activated sludge (in mineral medium4) for 7 days at the test temperature (the actual temperature: 20.0 – 21.8 °C). Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 10E-2, 10E-3, 10E-4 and 10E-5 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. During the pre-conditioning the approximately cell count of aerated inoculum was ~10E10 cells/L; therefore, before the test the inoculum was further diluted 1.000 000 x with mineral medium to reach the necessary 10E4 - 10E6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improved the precision of the test methods by reducing blank values. The inoculum was not pre-adapted to the test chemical.

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
In water (purified deionised) analytical grade salts were added to prepare the following stock solutions:

a) KH2PO4, 8.50 g
K2HPO4, 21.75 g
Na2HPO4 x 12H2O 67.16 g
NH4Cl 0.50 g
Water ad. 1000 mL

b) CaCl2 x 2H2O 36.40 g
Water ad. 1000 mL

c) MgSO4 x 7 H2O 22.50 g
Water ad. 1000 mL

d) FeCl3 x 6H2O 0.125 g
Water ad. 500 mL

1 mL of each of the stock solutions a - d) were combined and filled to a final volume of 1000 mL with deionised water (aqua purificata)3. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found to be 8.67 mg/L. The pH of the mineral medium was 7.38.

- Additional substrate: No
- Test temperature: 20.0 - 20.1 °C
- pH: 7.38
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Winkler bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aereation
- Measuring equipment: Oxygen and pH meter with appropriate O2 and pH electrode,
- Test performed in closed vessels

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum (12 mL) was added to the aqueous test medium (ad. 6000 mL).
- Toxicity control: Test (600 mL) and reference item (60 mL) stock solutions were mixed into the aqueous test medium (ad. 6000 mL) corresponding to the test item concentration of 3.0 mg/L [chosen based on the preliminary experiment and ThODNH4] and to 3.0 mg/L concentration of the reference item.
- Procedure Control: Based on the theoretical oxygen demand (ThODNH4) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), at the start of the test a stock solution of sodium benzoate was mixed into the aqueous test medium (corresponding to 3.0 mg/L reference item, respectively a ThODNH4 of about 5.01 mg O2/L).

STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The pre-experiments on solubility of the test item was not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of the present study.
In the pre-experiment the test item solubility was investigated in ultrapure water. At the concentration of 40 mg/L a homogeneous opalescent suspension was obtained after about 30 min. ultrasonic treatment.

Key result

Parameter:

% degradation (O2 consumption)

Value:

84.5

Sampling time:

28 d

Details on results:

Biodegradation of the Test Item
Under the test conditions the percentage biodegradation of the test item reached a mean of 84.5 % after 28 days based on its ThODNH4.
The number of bottles (8 measurement occasions) was applicable and allowed a relatively accurate estimation of the biodegradation processes. In this test the biodegradation curve reached its plateau on about the 12-14th days, from that days slight increases in biodegradability values occurred; however for informative reason the test was not stopped before the 28th day. The test item biodegradation of 71.8 % was above the pass level of 60 % of ThODNH4 already on the seventh day of the test consequently fulfilled the criterion for ready biodegradability since it surely reached the pass level of 60% of ThODNH4 in a 10-day window within the 28-d period of the test.

Biodegradation of Toxicity Control
In the toxicity control containing both, the test item and the reference item, a mean of 58.2 % biodegradation was noted within 14 days and a mean of 59.9 % biodegradation was determined after 28 days of incubation (from the 5th day of the test the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7% after 28 days of incubation, based on its ThODNH4. The biodegradability reached its plateau on about the 14th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Validity of the Study

Inoculum Control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.36 mg O2/L in average.

Oxygen Concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 0.73 mg O2/L, it was measured on the 28th day in the toxicity control.)

Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %. The highest difference (13.7 %) between the duplicate values from the 5th to the 28th day of the test (this period was taken into consideration as biodegradation plateau) or at the end of the test (test item, procedure control, and toxicity control groups) for degradation was calculated in the procedure control group, it was observed on the 7th day of the test.

Reference Item: The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 84.0 % on the 14th day.)

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In a biodegradability screening test according to OECD guideline 301 D, the test item is considered to be ready biodegradable, since it fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window.

Executive summary:

The biodegradability of the test item was determined in a screening test according to OECD guideline 301D. The test item was exposed to activated sludge (non-adapted) from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 3.0 mg/L.

The test item concentration was chosen based on its theoretical oxygen demand (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.22 mg O2/mg.

In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met.

The percentage biodegradation ofthe test itemreached a mean of 84.5 % after 28 days based on its ThODNH4. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, more than 60 % biodegradation of the test item (71.8 %, based on its ThODNH4) was observed on the seventh day of the test. The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 14th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 58.2 % biodegradation was noted within 14 days and 59.9 % biodegradation after 28 days of incubation (from the 5th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

The test item is considered to be ready biodegradable, since it fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window.

Description of key information

In a biodegradability screening test according to OECD guideline 301 D, the test item is considered to be ready biodegradable, since it fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The biodegradability of the test item was determined in a screening test according to OECD guideline 301D. The test item was exposed to activated sludge (non-adapted) from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 3.0 mg/L.

The test item concentration was chosen based on its theoretical oxygen demand (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.22 mg O2/mg.

In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met.

The percentage biodegradation ofthe test itemreached a mean of 84.5 % after 28 days based on its ThODNH4. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, more than 60 % biodegradation of the test item (71.8 %, based on its ThODNH4) was observed on the seventh day of the test. The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 14th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 58.2 % biodegradation was noted within 14 days and 59.9 % biodegradation after 28 days of incubation (from the 5th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

The test item is considered to be ready biodegradable, since it fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window.