Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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 2007-11-09 to 2007-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Test System
Species: Activated sludge, microorganisms from a domestic waste water treatment plant
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: 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 4 g dry material per litre were mixed with test water and then aerated until use. Before use the sludge was filtered through cotton wool.

Duration of test (contact time):

28 d

Initial conc.:

1.5 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Units
Type and Size: BOD bottles of 300 mL volume with special neck and glass stoppers.
Identification: Each of the test flasks were uniquely identified with study code, test group, days of measurement and replicate number.

Test Conditions
Surrounding Type: Incubator and controlled environment room (during the formulation and oxygen measuring)
Temperature: 19.6 - 20.7 °C
pH-Value of Reconstituted Water: 7.23 (measured at the start of the test)
Oxygen conc. of Reconstituted Water: 9.0 mg O2/L (measured at the start of the test)
Reconstituted Water: In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 4.25 g KH2PO4, 10.875 g K2HPO4, 33.59 g Na2HPO4 x 12H2O, 0.25 g NH4Cl filled up with deionised water to 500 mL volume
b) 11.25 g MgSO4 x 7H2O filled up with deionised water to 500 mL volume
c) 18.20 g CaCl2 x 2H2O filled up with deionised water to 500 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume

Ratio of ingredients: 1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. 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 about 9.0 mg/L at about 20 °C.

Equipments:
Large glass tanks with valve,
Large glass bottles with valve,
Narrow necked, 300 mL BOD bottles with glass stoppers,
Funnels and coarse filter papers,
Moisture Analyzer,
Centrifuge,
Balance,
Self stirring O2 electrode,
Oxygen and pH meter,
Aeration system,
Incubator with thermometer

Study Design
Preparation of the Test Solutions
The components were applied in the following ratio in the test flasks:
The preparation of the respective test solutions with the test item was performed according to the followings:
The respective amount of the test item was weighed in directly to reach the required test item concentration of 1.5 mg/L. During the performance of the test the test solutions were mixed e.g. by mechanical stirring to ensure a good dispersion.

Test Item (flasks 1a and 1b):
Based on the theoretical oxygen demand (ThODNO3) of 2.87 mg O2/mg test item (calculated according to equation given in the guidelines), 10.38 mg of the test item was thoroughly mixed into 6.92 litres of aqueous test medium (corresponding to 1.5 mg/L test item, respectively a ThODNO3 of about 4.305 mg O2/L).

Procedure Control: Sodium acetate (flasks 2a and 2b):
Based on the theoretical oxygen demand (ThODNH4) of Sodium acetate (0.78 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 51.072 mg of Sodium acetate was mixed into 6.72 litres of aqueous test medium (corresponding to 7.6 mg/L reference item, respectively a ThODNH4 of about 5.93 mg O2/L).
* the concentration of the stock solution was 760 mg/L

Inoculum Control (flasks 3a and 3b):
Only filtered inoculum was added to 6.80 litres of aqueous test medium.

Toxicity Control (flasks 4a and 4b):
10.38 mg of Reaction Product of Bisphenol A (BADGE) with IPDA and reference item stock solution* (69.2 mL) were mixed into 6.92 litres of aqueous test medium corresponding to 1.5 mg/L test item (ThODNO3 of 4.305 mg O2/L) and 7.6 mg/L reference item (ThODNH4 of 5.93 mg O2/L).
* the concentration of the stock solution was 760 mg/L

General: Microbial inoculum (2.0 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 stoppered.

The following bottles were prepared:
10 (+2 reserve) bottles containing the test item and inoculum
16 (+2 reserve) bottles containing the reference item and inoculum (procedure control) *
16 (+2 reserve) bottles containing only inoculum (inoculum control) *
10 (+2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
* Remark: Because of a parallel running study at the inoculum and procedure control 16 (+2 reserve) bottles were prepared instead of 10 (+2 reserve).

Incubation Period: 28 days

Test Parameters
Measurement of Oxygen: The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode.
Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28 *.
* Remark: Because of a parallel running study at the inoculum and procedure control oxygen measurements were performed on days: 0, 2, 5, 7, 12, 14, 21 and 28; however the measured oxygen concentration values on days: 2, 5, 12 were not used in this study.

Measurement of total oxidised N: (nitrite and nitrate):
Because of 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 total oxidised nitrogen (nitrate and nitrite) concentrations were determined directly after each oxygen measurement.
The sum of the nitrate and nitrite content was measured with HPLC ion exchange method. The samples were injected directly.

Measurement of Temperature:
Temperature was measured continuously and registered on weekdays.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

2.3

Sampling time:

28 d

Details on results:

Correction for oxygen uptake for interference with nitrification:
Because of the N-containing test item, the total oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement. The total oxidised nitrogen content was calculated referring to the sum of non-oxidised nitrogen (N mg/L) content of the samples. The LOQ of nitrite and nitrate measurements was 0.05 μg NO2/mL and 0.05 μg NO3/mL, respectively. The measured quantity of total oxidised nitrogen (nitrite and nitrate) was below the LOQ in the measured samples during the experiment. The total oxidised nitrogen concentration was below the LOQ in the measured samples (samples of test item, inoculum control and toxicity control group) during the experiment. Therefore, the calculated BOD values were not corrected for nitrification.

Percentage Biodegradation:
Under the test conditions the percentage biodegradation of the test item reached a mean of 2.3 % after 28 days based on ThODNO3. The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.

Key result

Parameter:

BOD5

Value:

>= -0.03 - <= 0.13 other: mgO2/mg test. mat.

Results with reference substance:

The reference item Sodium acetate was sufficiently degraded to a mean of 71.6 % after 14 days, and to a mean of 78.4 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Biodegradation of Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 26.2 % biodegradation was noted within 14 days and a mean of 27.1 % biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.

Executive summary:

The purpose of this study was to investigate the ready biodegradability of the test item 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 acetate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Under the test conditions the percentage biodegradation of the test item reached a mean of 2.3 % after 28 days based on ThODNO3. Therefore the test item can be considered to be not ready biodegradable.

The reference item Sodium acetate was sufficiently degraded to a mean of 71.6 % after 14 days, and to a mean of 78.4 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 26.2 % biodegradation was noted within 14 days and 27.1 % biodegradation after 28 days of incubation.

Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Description of key information

The ready biodegradability of the test item was studied in a closed bottle test. The test item was exposed to activated sludge for 28 days. Under test conditions the percentage biodegradation of the test item reached a mean of 2.3 % after 28 days based on the ThODNO3.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The purpose of this study was to investigate the ready biodegradability of the test item 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 acetate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Under the test conditions the percentage biodegradation of the test item reached a mean of 2.3 % after 28 days based on ThODNO3. Therefore the test item can be considered to be not ready biodegradable.

The reference item Sodium acetate was sufficiently degraded to a mean of 71.6 % after 14 days, and to a mean of 78.4 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 26.2 % biodegradation was noted within 14 days and 27.1 % biodegradation after 28 days of incubation.

Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).