Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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 2016-05-18 to 2016-08-17

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:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 12 May 2016 (7 days before the test).
- Storage conditions: Tightly closed containers kept in a dry, room temperature and well-ventilated place.
- 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 and then aerated under test conditions (for 7 days) until use.
- Pretreatment: No pretreatment was performed (the inoculum was not pre-adapted to the test chemical).
- Pre-conditioning: Pre-conditioning was performed to improve the precision of the test methods by reducing blank values. Pre-conditioning (12-19 May 2016) consisted of aerating (2 L/minute) activated sludge (in mineral medium, reconstituted water) for 7 days at the test temperature (the actual temperature range was 20.6 - 22.0 °C).
- Concentration of sludge: 5 g dry material/L
- Initial cell/biomass concentration: The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of 10E8 – 10E9/L; therefore on the day of the test this inoculum was diluted adequately with reconstituted water to reach the necessary cell concentration.
- Water filtered: Yes
- Type and size of filter used: Cotton wool

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Initial conc.:

2.55 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The composition of the test medium was performed according to the OECD guideline.
- Additional substrate: No
- Solubilising agent: Not used
- Test temperature: During the preparation, aeration and incubation of the reconstituted water the temperature was 20.3 - 21.1 °C.
During the incubation (28 days) of the test units the temperature range was the following: 20.0 - 20.5 °C.
- pH value of the test water: The pH was checked prior study start and found to be 7.34.
- pH of the activated sludge inoculum after preparation was 7.01, just before use: 7.27.
- pH adjusted: No, pH adjustment was considered not necessary.
- Aeration of dilution water: Yes
- Suspended solids concentration: 5 g dry material per litre
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers
- Number of culture flasks/concentration:
10 (+2 reserve) bottles containing the test item and inoculum
10 (+2 reserve) bottles containing the reference item and inoculum (procedure control)
10 (+2 reserve) bottles containing only inoculum (inoculum control)
10 (+2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
- Method used to create aerobic conditions: The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature.
- Measuring equipment:
Large glass bottles (volume: 5 L),
Narrow necked, Winkler bottles with glass stoppers,
Funnels and coarse filter papers,
Oxygen and pH meter with appropriate O2 and pH electrode,
Aeration system, Moisture analyser
Temperature controlled (22 ± 2 °C) environment room and incubator with thermometer with exclusion of light,
Balance, Centrifuge
- Measurement of Oxygen: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
- Measurement of Chemical Oxygen Demand (COD): There is no available information about the test item molecular formula; therefore the COD value of the test item was determined using a COD Cell Test (MERCK) and CombiCheck 50.
- Measurement of total oxidized N: (nitrite and nitrate): Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidized nitrogen (nitrate and nitrite) concentrations were measured.
- Measurement of temperature: Temperature was measured continuously using a built-in thermometer of the thermostat and noticed once or twice a day.

SAMPLING
No concentrations of the test substance were determined. Determination of the nitrate and nitrite ion concentrations in test solutions after the dissolved oxygen concentration measurements were performed on day 0, and on the 7th, 14th, 21st and 28th day of the test.
- Samples: Nitrate and nitrite ion concentrations in the test solutions were determined on the 7th, 21st and 28th day of the test.
- Analytical Method: The nitrite and nitrate concentrations were determined using photometric method with nitrite and nitrate cell test (Merck).
- Nitrate concentration in the test solutions: The nitrate concentration of the samples was less than 0.4 mg/L on the 0, 7th, 14th, 21st and on 28th day samples.
- Nitrite concentration in the test solutions: The nitrite concentration of the samples was less than 0.03 mg/L on the 0, 7th, 14th, 21st and on 28th day samples.
- Samples storage: The taken samples were stored in freezer until measurements of nitrate and nitrite ion concentrations.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. Only filtered inoculum was added to the aqueous test medium without the test substance.
- Abiotic sterile control: Not performed.
- Toxicity control: Yes. It was performed with the test item, reference item and inoculum.
- Procedure Control: Yes. Performed with sodium benzoate

Reference substance:

benzoic acid, sodium salt

Preliminary study:

A preliminary test based on the measured chemical oxygen demand (COD) of the test substance was performed to determine the concentration of the test substance. The COD value was determined to be 2.55 mgO2/mg test item.

Test performance:

The validity criteria were met:
- Inoculum control; The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.2 mg O2/L in average
- Oxygen concentration: The residual oxygen concentration in the test flasks did not drop below 0.5 mg O2/L at any time. (The lowest value was 0.55 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 (12.7 %) between the duplicate values from the 14th 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 21st day.
- 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 67.8 % on the 14th day.

Key result

Parameter:

% degradation (O2 consumption)

Value:

47.4

Sampling time:

28 d

Details on results:

The percentage biodegradation of the test substance reached a mean of 47.4% after 28 days based on its COD.The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item was calculated based on its COD; any correction, based on the nitrite and/or nitrate content was not performed.
In the toxicity control containing both, the test item and the reference item, a mean of 41.9 % biodegradation was noted within 14 days and 50.5 % biodegradation after 28 days of incubation.
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 percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum but the test item is considered to be not ready biodegradable, since the pass level for ready biodegradability is removal of 60% COD in a 10-day window.

Results with reference substance:

The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. The reference item sodium benzoate was sufficiently degraded to a mean of 67.8 % after 14 days, and to a mean of 78.1 % after 28 days of incubation, based on ThODNH4.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

The ready biodegradability of the test substance was investigated according to the OECD 301D: Closed bottle test (1992). The percentage of the biodegradation of the test substance reached a mean of 47.4 % after 28 days based on its COD. The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability is removal of 60% COD. Nevertheless, the substance can be considered to be inherently biodegradable as a mean biodegradation of 47.4 % after 28 days was observed.

Executive summary:

The ready biodegradability of the test substance was investigated according to the OECD guideline 301D: Closed bottle test (1992) and according to Council Directive 92/69 EEC, Method CA-E (1992) under GLP conditions. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant 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 concentration was chosen based on the preliminary test results and based on the measured chemical oxygen demand (COD) of 2.55 mg O2/mg test item. 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. The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item was calculated based on its COD; any correction, based on the nitrite and/or nitrate content was not performed. The reference item sodium benzoate was sufficiently degraded to a mean of 67.8 % after 14 days, and to a mean of 78.1 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 41.9 % biodegradation was noted within 14 days and 50.5 % biodegradation after 28 days of incubation. 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 percentage biodegradation of the test substance reached a mean of 47.4% after 28 days based on its COD. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. All validity criteria of the study were met. The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability is removal of 60 % COD. Nevertheless, the substance can be considered to be inherently biodegradable as a mean biodegradation of 47.4 % after 28 days was observed.

Description of key information

The ready biodegradability of the test substance was investigated according to the OECD 301D: Closed bottle test (1992). The percentage of the biodegradation of the test substance reached a mean of 47.4 % after 28 days based on its COD. The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability is removal of 60% COD. Nevertheless, the substance can be considered to be inherently biodegradable as a mean biodegradation of 47.4 % after 28 days was observed.

.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Additional information

The ready biodegradability of the test substance was investigated according to the OECD guideline 301D: Closed bottle test (1992) and according to Council Directive 92/69 EEC, Method CA-E (1992) under GLP conditions. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant 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 concentration was chosen based on the preliminary test results and based on the measured chemical oxygen demand (COD) of 2.55 mg O2/mg test item. 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. The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item was calculated based on its COD; any correction, based on the nitrite and/or nitrate content was not performed. The reference item sodium benzoate was sufficiently degraded to a mean of 67.8 % after 14 days, and to a mean of 78.1 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 41.9 % biodegradation was noted within 14 days and 50.5 % biodegradation after 28 days of incubation. 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 percentage biodegradation of the test substance reached a mean of 47.4% after 28 days based on its COD. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability is removal of 60 % COD in a 10-day window. All validity criteria of the study were met.